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Made available in DSpace on 2022-07-15T10:20:15Z (GMT). No. of bitstreams: 1 Plant-Pathology-2022-Berbert-Expression-of-a-sphingomyelinase8208coding-gene-from-Trichoderma-harzianum-conferred.pdf: 9944005 bytes, checksum: 79c1bd418d7784469654676001102e52 (MD5) Previous issue date: 2022

Huanglongbing (HLB) has been a serious threat to the citrus industry worldwide. After its first report in Sao Paulo State, the main citrus production area in Brazil, the disease spread to the States of Minas Gerais, Parana and Mato Grosso do Sul. Attempts to cure plants or remiss disease symptoms and damages have been evaluated and showed to be inefficient and nonviable. The development of resistant or tolerant varieties to the bacteria or its insect vector, the Asian citrus psyllid Diaphorina citri, is still a long term challenge. Earlier HLB management has been based on preventive measures such as planting of healthy nursery trees, elimination of diseased trees, and vector control. Supported by both research data and citrus grower experiences, HLB management in Sao Paulo and Triângulo/Sudoeste Mineiro citrus belt has been improved from measures individually applied only into the orchards to regional disease management, including differentiated psyllid control in the orchards based on tree location and shoot flushing, area-wide coordinated control of psyllids, and removal of inoculum sources in noncommercial properties in the vicinity of commercial orchards. In addition, the negative impact of HLB on orchard production and longevity has been reduced with wide adoption of better cultural practices such as high-density planting, irrigation, and adequate nutrition. Unlike in other countries where HLB reached epidemic levels, the management of HLB in Sao Paulo and Triângulo/Sudoeste Mineiro citrus belt has been considered a success case and has ensured the maintenance of citrus production and competitiveness of the Brazilian citrus industry while new, more durable, and sustainable measures are not yet available.

Citrus plants presenting the typical huanglongbing (HLB) symptoms have been associated to 16SrIX phytoplasma in the States of Sao Paulo (SP), Minas Gerais (MG), Bahia (BA) and, more recently, Distrito Federal (DF), Brazil. Although in SP and MG states ‘Candidatus Liberibacter asiaticus’ is more commonly associated with HLB, single infection by phytoplasmas also occurs. To date, only 16SrIX phytoplasma induces HLB symptoms in BA and DF, a region free of ‘Ca. Liberibacter spp.’. The objective of this work was to survey for alternative hosts and potential vectors of the 16SrIX phytoplasma in citrus orchards from DF using quantitative real-time PCR (qPCR) detection. Fifty-three specimens encompassing fifteen species of weeds were collected from three Tahiti lime (Citrus latifolia Tanaka) and Ponkan mandarin (Citrus reticulata Blanco) orchards. Only five plants, one Euphorbia sp., one Sida sp., and three Bidens pilosa were positive for the presence of the 16SrIX phytoplasma. Regarding the leafhoppers, 16SrIX phytoplasma was detected in 11/ 38 specimens of Scaphytopius sp. and not detected in 32 specimens of Planicephalus flavicosta. These results suggest that spontaneous vegetation and the leafhoppers Scaphytopius may harbor 16SrIX phytoplasma that can further infect citrus orchards. The capacity of Scaphytopius insects to vector the phytoplasma to citrus plant should be better investigated.

Submitted by Maria Cândida de Godoy Gasparoto (maria.candida@unesp.br) on 2021-08-20T13:44:00Z No. of bitstreams: 1 Gasparoto et al 2021 First Look - Repositório.pdf: 2550316 bytes, checksum: 24271d4e581fde04f2da07713c13d3e7 (MD5) Approved for entry into archive by Luana Priscila Costa (luana@reitoria.unesp.br) on 2021-09-15T19:12:52Z (GMT) No. of bitstreams: 1 gasparoto_mc_preprint_regi_prevalent.pdf: 2550316 bytes, checksum: 24271d4e581fde04f2da07713c13d3e7 (MD5) Made available in DSpace on 2021-09-15T19:12:52Z (GMT). No. of bitstreams: 1 gasparoto_mc_preprint_regi_prevalent.pdf: 2550316 bytes, checksum: 24271d4e581fde04f2da07713c13d3e7 (MD5) Previous issue date: 2021-08-19 Outra Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) In Brazil, citrus huanglongbing (HLB) is associated with ‘Candidatus Liberibacter americanus’ (CLam) and ‘Ca. Liberibacter asiaticus’ (CLas). However, there are few studies about HLB epidemiology when both Liberibacter spp. and its insect vector, the Asian citrus psyllid (ACP, Diaphorina citri), are present. The objective of this work was to compare the transmission of HLB by ACP when both CLam and CLas are present as primary inoculum. Two experiments were performed under screenhouse conditions from April 2008 to January 2012 (experiment 1) and from February 2011 to December 2015 (experiment 2). The experiments were carried out with sweet orange plants infected with CLam or CLas as inoculum source surrounded by sweet orange healthy plants. One hundred Liberibacter-free adult psyllids were monthly confined to the source of inoculum plants for 7 days with subsequent free movement inside the screenhouse. Fortnightly, nymphs and adults of psyllids were monitored. Psyllid and leaf samples were collected periodically for Liberibacter detection by PCR or qPCR. CLas was detected more frequently than CLam in both psyllid and leaf samples. No mixed infections were detected in the psyllids. A clear prevalence of CLas over CLam was observed in both experiments. The final HLB incidences were 16.7 and 14.5% of Liberibacter-positive test plants, and CLas was detected in 92.3 and 93.1% of these infected plants. Mixed infection was observed only in 3.8% of infected test plants in experiment 1. These results endorse the shift in the prevalence of CLam to CLas observed in citrus orchards of São Paulo, Brazil. Postprint Universidade Estadual Paulista "Júlio de Mesquita Filho" (Unesp) Fundo de Defesa da Citricultura (Fundecitrus) Universidade de São Paulo (Usp) FAPESP: 2007/55013-9 FAPESP: 2007/52040-5 FUNDECITRUS: 043012 Citrus Research Development Foundation: NAS7

In Brazil, citrus huanglongbing (HLB) is associated with '

‘Candidatus Liberibacter asiaticus’, an unculturable α-proteobacterium, is associated with citrus huanglongbing (HLB), a devastating disease threatening citrus production in Brazil and worldwide. In this study, a draft whole-genome sequence of ‘Ca. L. asiaticus’ strain 9PA from a sweet orange (cultivar Pera) tree collected in São Paulo State, Brazil, is reported. The 9PA genome is 1,231,881 bp, including two prophages, with G+C content of 36.7%. This is the first report of a whole-genome sequence of ‘Ca. L. asiaticus’ from Brazil or South America. The 9PA genome sequence will enrich ‘Ca. L. asiaticus’ genome resources and facilitate HLB research and control in Brazil and the world.

Made available in DSpace on 2022-04-29T08:45:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-07-12 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The Asian citrus psyllid, Diaphorina citri, is the vector of the bacterium “Candidatus Liberibacter asiaticus” (Las), associated with the devastating, worldwide citrus disease huanglongbing. In order to explore the molecular interactions of this bacterium with D. citri during the vector acquisition process, cDNA libraries were sequenced on an Illumina platform, obtained from the gut of adult psyllids confined in healthy (H) and in Las-infected young shoots (Las) for different periods of times (I = 1/2 days, II = 3/4 days, and III = 5/6 days). In each sampling time, three biological replicates were collected, containing 100 guts each, totaling 18 libraries depleted in ribosomal RNA. Reads were quality-filtered and mapped against the Chinese JXGC Las strain and the Floridian strain UF506 for the analysis of the activity of Las genome and SC1, SC2, and type 3 (P-JXGC-3) prophages of the studied Las strain. Gene activity was considered only if reads of at least two replicates for each acquisition access period mapped against the selected genomes, which resulted in coverages of 44.4, 79.9, and 94.5% of the JXGC predicted coding sequences in Las I, Las II, and Las III, respectively. These genes indicate an active metabolism and increased expression according to the feeding time in the following functional categories: energy production, amino acid metabolism, signal translation, cell wall, and replication and repair of genetic material. Pilins were among the most highly expressed genes regardless of the acquisition time, while only a few genes from cluster I of flagella were not expressed. Furthermore, the prophage region had a greater coverage of reads for SC1 and P-JXGC-3 prophages and low coverage in SC2 and no indication of activity for the lysis cycle. This research presents the first descriptive analysis of Las transcriptome in the initial steps of the D. citri gut colonization, where 95% of Las genes were active. Instituto de Química Universidade Estadual Paulista “Julio de Mesquita Filho” – UNESP Departamento de Pesquisa Desenvolvimento Fundo de Defesa da Citricultura – Fundecitrus Laboratório de Interações em Insetos Departamento de Entomologia e Acarologia Escola Superior de Agricultura Luiz de Queiroz Universidade de São Paulo Instituto de Biologia Molecular y Celular de Plantas – Consejo Superior de Investigaciones Científicas Universidade Politécnica de Valencia Instituto de Química Universidade Estadual Paulista “Julio de Mesquita Filho” – UNESP

‘Candidatus Liberibacter asiaticus’ is the most common huanglongbing-associated bacteria, being present in Asia, South, Central, and North America. Genomic approaches enabled sequencing of ‘Ca. L. asiaticus’ genomes, allowing for a broader assessment of its genetic variability with the application of polymerase chain reaction (PCR)-based tools such as microsatellite or short tandem repeat (STR) analysis. Although these tools contributed to a detailed analysis of strains from Japan, China, and the United States, Brazilian strains were analyzed in either too few samples with several STRs or in several strains with only a single microsatellite and a single PCR marker. We used 573 ‘Ca. L. asiaticus’ strains, mainly collected from São Paulo State (SPS), in our genetic analyses, employing three STRs and several prophage PCR markers. STR revealed a homogeneous population regardless of sampling year or geographic regions of SPS. Thirty-eight haplotypes were recognized with a predominance of VNTR_005 higher than 10 repeats, with VNTR_002 and VNTR_077 containing 11 and 8 repeats, respectively. This haplotype is indicated as class HE, which comprised 80.28% of strains. Classes HA and HB, predominant in Florida, were not found. A new genomic organization in the junction of prophages SC2 and SC1 is prevalent in Brazilian strains, indicating gene rearrangement and a widespread occurrence of a type 1 prophage as well as the presence of a type 2-like prophage. Our results indicate that ‘Ca. L. asiaticus’ populations are homogeneous and harbor a new genomic organization in prophages type 1 and 2.

The Asian citrus psyllid, Diaphorina citri, is the vector of the bacterium “Candidatus Liberibacter asiaticus” (Las), associated with the devastating, worldwide citrus disease huanglongbing. In order to explore the molecular interactions of this bacterium with D. citri during the vector acquisition process, cDNA libraries were sequenced on an Illumina platform, obtained from the gut of adult psyllids confined in healthy (H) and in Las-infected young shoots (Las) for different periods of times (I = 1/2 days, II = 3/4 days, and III = 5/6 days). In each sampling time, three biological replicates were collected, containing 100 guts each, totaling 18 libraries depleted in ribosomal RNA. Reads were quality-filtered and mapped against the Chinese JXGC Las strain and the Floridian strain UF506 for the analysis of the activity of Las genome and SC1, SC2, and type 3 (P-JXGC-3) prophages of the studied Las strain. Gene activity was considered only if reads of at least two replicates for each acquisition access period mapped against the selected genomes, which resulted in coverages of 44.4, 79.9, and 94.5% of the JXGC predicted coding sequences in Las I, Las II, and Las III, respectively. These genes indicate an active metabolism and increased expression according to the feeding time in the following functional categories: energy production, amino acid metabolism, signal translation, cell wall, and replication and repair of genetic material. Pilins were among the most highly expressed genes regardless of the acquisition time, while only a few genes from cluster I of flagella were not expressed. Furthermore, the prophage region had a greater coverage of reads for SC1 and P-JXGC-3 prophages and low coverage in SC2 and no indication of activity for the lysis cycle. This research presents the first descriptive analysis of Las transcriptome in the initial steps of the D. citri gut colonization, where 95% of Las genes were active.

[EN] Huanglongbing (HLB) is a destructive disease, associated with psyllid-transmitted phloem-restricted pathogenic bacteria, which is seriously endangering citriculture worldwide. It affects all citrus species and cultivars regardless of the rootstock used, and despite intensive research in the last decades, there is no effective cure to control either the bacterial species (Candidatus Liberibacter spp.) or their insect vectors (Diaphorina citri and Trioza erytreae). Currently, the best attempts to manage HLB are based on three approaches: (i) reducing the psyllid population by intensive insecticide treatments; (ii) reducing inoculum sources by removing infected trees, and (iii) using nursery-certified healthy plants for replanting. The economic losses caused by HLB (decreased fruit quality, reduced yield, and tree destruction) and the huge environmental costs of disease management seriously threaten the sustainability of the citrus industry in affected regions. Here, we have generated genetically modified sweet orange lines to constitutively emit (E)-beta-caryophyllene, a sesquiterpene repellent to D. citri, the main HLB psyllid vector. We demonstrate that this alteration in volatile emission affects behavioral responses of the psyllid in olfactometric and no-choice assays, making them repellent/less attractant to the HLB vector, opening a new alternative for possible HLB control in the field., This work was funded by the Fundo de Defesa da Citricultura (Fundecitrus), Sao Paulo Research Foundation (FAPESP, grant #2015/07011-3), and EU H2020 Innovation Action Program (grant #817526). Consent for research and field trial of genetically modified organisms was granted by the National Technical Biosafety Commission from Brazil (CTNBio) to Fundecitrus.

Candidatus Liberibacter asiaticus (CLas) is one the causative agents of greening disease in citrus, an unccurable, devastating disease of citrus worldwide. CLas is vectored by Diaphorina citri, and the understanding of the molecular interplay between vector and pathogen will provide additional basis for the development and implementation of successful management strategies. We focused in the molecular interplay occurring in the gut of the vector, a major barrier for CLas invasion and colonization. We investigated the differential expression of vector and CLas genes by analyzing a de novo reference metatranscriptome of the gut of adult psyllids fed of CLas-infected and healthy citrus plants for 1-2, 3-4 and 5-6 days. CLas regulates the immune response of the vector affecting the production of reactive species of oxygen and nitrogen, and the production of antimicrobial peptides. Moreover, CLas overexpressed peroxiredoxin in a protective manner. The major transcript involved in immune expression was related to melanization, a CLIP-domain serine protease we believe participates in the wounding of epithelial cells damaged during infection, which is supported by the down-regulation of pangolin. We also detected that CLas modulates the gut peristalsis of psyllids through the down-regulation of titin, reducing the elimination of CLas with faeces. The up-regulation of the neuromodulator arylalkylamine N-acetyltransferase implies CLas also interferes with the double brain-gut communication circuitry of the vector. CLas colonizes the gut by expressing two Type IVb pilin flp genes and several chaperones that can also function as adhesins. We hypothesized biofil formation occurs by the expression of the cold shock protein of CLas. We also describe the interplay during cell invasion and modification, and propose mechanisms CLas uses to invade the host hemocel. We identified several specific targets for the development of strategies directed to interfere with the successful utilization of the psyllid vector by this pathogen.Author SummaryHuanglongbing (HLB) or greening is an incurable disease causing severe damage to citrus production, making citrus industrial activity unsustainable in several countries around the world. HLB is caused by three species of Candidatus Liberibacter. Ca. L. asiaticus (CLas), vectored by the psyllid Diaphorina citri, is the prevalent species. Attempts to apply new technologies in the development of strategies for disease and pest management are been made. However, we still miss basic information on this system to efficiently apply the current technologies and envisage the implementation of new approaches for pest control, despite the relevant scientific contribution available. One major gap is regarded to the molecular interplay between CLas and its vector. We focused our attention in the molecular interplay occurring at the first relevant interaction of CLas and D. citri, represented by the gut barrier. We report the transcriptional activity of CLas during the invasion and establishment of the infection in the gut of the vector, as well as the transcriptional activity of the vector in response to the infection. We identified several host genes that are targeted and regulated by CLas as well as several CLas genes that are promising targets for the application of new management strategies.

Huanglongbing (HLB) is a destructive disease, associated with psyllid-transmitted phloem-restricted pathogenic bacteria, which is seriously endangering citriculture worldwide. It affects all citrus species and cultivars regardless of the rootstock used, and despite intensive research in the last decades, there is no effective cure to control either the bacterial species (Candidatus Liberibacter spp.) or their insect vectors (Diaphorina citri and Trioza erytreae). Currently, the best attempts to manage HLB are based on three approaches: (i) reducing the psyllid population by intensive insecticide treatments; (ii) reducing inoculum sources by removing infected trees, and (iii) using nursery-certified healthy plants for replanting. The economic losses caused by HLB (decreased fruit quality, reduced yield, and tree destruction) and the huge environmental costs of disease management seriously threaten the sustainability of the citrus industry in affected regions. Here, we have generated genetically modified sweet orange lines to constitutively emit (E)-β-caryophyllene, a sesquiterpene repellent to D. citri, the main HLB psyllid vector. We demonstrate that this alteration in volatile emission affects behavioral responses of the psyllid in olfactometric and no-choice assays, making them repellent/less attractant to the HLB vector, opening a new alternative for possible HLB control in the field.

Simple Summary Candidatus Liberibacter spp. associated with citrus huanglongbing (HLB) comprise species Ca. L. africanus (Laf), Ca. L. asiaticus (Las), and Ca. L. americanus (Lam). While Laf is vectored by the psyllid Trioza erytreae, Las and Lam are vectored by Diaphorina citri. Las spread by D. citri is a major factor for the incidence of HLB-affected citrus in Asia and South, Central, and North America. Recently, both D. citri and Las were also detected in continental Africa, while T. erytrea was found in Portugal and Spain. D. citri that feed as nymphs in citrus harboring Las are better vectors when become adults than adults that acquire Las. Planting nursery trees free of Ca. Liberibacter spp., removing diseased symptomatic citrus trees, and controlling the psyllid population are management practices able to keep infection at acceptable rates. Psyllid population levels, as well as the percentage of individuals that carry Liberibacter, are important factors to succeed against HLB. Area-wide HLB management has been used in Brazil since 2009 and, in this context, psyllid population increase is monitored for control. The same integrated system was used to collect psyllids from traps over a three-year period, as a large-scale resource to address the effect of local management on the percentage of D. citri harboring Las. Good HLB management practices resulted in lower numbers of psyllids with Las. Location and regional disease incidence also affected the percentage of psyllids with Las, ranging from 33 to 74.6%. Abstract Huanglongbing (HLB) is a citrus disease of worldwide importance, associated with the presence of Candidatus Liberibacter asiaticus (Las) and vectored by the psyllid Diaphorina citri in Asia and the Americas. To properly manage HLB, removal of inoculum sources and control of the psyllid are undertaken. We evaluated the percentage of the psyllid population with Las, sampled from yellow sticky traps over a three-year period and its relationship with insect population, regions, season of the year, and HLB management in citrus areas in the southwestern, central, and northern regions of São Paulo (SP) and southwestern region of Minas Gerais states, Brazil. In each reading, up to 50 psyllids per region were collected and detection of Las in individual psyllids were made by quantitative polymerase chain reaction. The percentage of psyllids with Las—an average of 65.3%—was constant throughout the year in the southwestern region of SP state, while showing an increase from spring to autumn when sampled from central to northern regions. The proportion of psyllids carrying Las from each region and year period were compared by a proportion test and spectral density analysis. The proportion of psyllids carrying Las evaluated in the same region in different seasons presented statistical differences in central (Araraquara) and southwestern (Santa Cruz do Rio Pardo) regions in 2015, with higher values in the first semester (summer and autumn) than in the second semester (winter and spring). Orchards with poor HLB management had higher incidence of psyllids with Las. Spectral density analysis indicated that good management areas had 50% less relevant peaks of psyllids with Las than in areas with poor HLB management practices. The relationship between the percentage of psyllids carrying Las and the number of captured psyllids in the region in a given time denotes the most critical intake time for HLB spread in citrus orchards. The reduction in the population of psyllids carrying Las is a direct benefit from the use of good management practices.

Candidatus Liberibacter asiaticus (CLas) is a phloem-limited bacterium that is associated with the Huanglongbing (HLB) disease of citrus and transmitted by the psyllid, Diaphorina citri. There are no curative methods to control HLB and the prevention of new infections is essential for HLB management. Therefore, the objective of our study was to determine the effects of systemic insecticides, such as the neonicotinoids imidacloprid, thiamethoxam, and a mixture of thiamethoxam and chlorantraniliprole (diamide) on the probing behavior of CLas-infected D. citri and their effect on CLas transmission. The electrical penetration graph (EPG-DC) technique was used to monitor the stylet penetration activities of CLas-infected D. citri on sweet orange [Citrus sinensis (L.) Osbeck] &lsquo, Valencia&rsquo, treated with systemic insecticides. Systemic insecticides disrupted the probing behavior of CLas-infected D. citri, in a way that affected CLas transmission efficiency, particularly by negatively affecting the stylet activities related to the phloem phase. All insecticides reduced (by 57&ndash, 73%) the proportion of psyllids that exhibited sustainable phloem ingestion (waveform E2 &gt, 10 min), with significant differences observed on plants treated with thiamethoxam and thiamethoxam + chlorantraniliprole. The transmission rate of CLas with high inoculum pressure (five CLas-infected D. citri per plant and a seven-day inoculation access period) to untreated control plants was 93%. In contrast, CLas transmission was reduced to 38.8% when test plants were protected by systemic insecticides. Our results indicated that all insecticides tested presented a potential to reduce CLas inoculation by an average of 59%, therefore, these insecticides can be used to reduce the spread of HLB.

Candidatus Liberibacter asiaticus (CLas) is a phloem-limited bacterium that is associated with the Huanglongbing (HLB) disease of citrus and transmitted by the psyllid, Diaphorina citri. There are no curative methods to control HLB and the prevention of new infections is essential for HLB management. Therefore, the objective of our study was to determine the effects of systemic insecticides, such as the neonicotinoids imidacloprid, thiamethoxam, and a mixture of thiamethoxam and chlorantraniliprole (diamide) on the probing behavior of CLas-infected D. citri and their effect on CLas transmission. The electrical penetration graph (EPG-DC) technique was used to monitor the stylet penetration activities of CLas-infected D. citri on sweet orange [Citrus sinensis (L.) Osbeck] ‘Valencia’ treated with systemic insecticides. Systemic insecticides disrupted the probing behavior of CLas-infected D. citri, in a way that affected CLas transmission efficiency, particularly by negatively affecting the stylet activities related to the phloem phase. All insecticides reduced (by 57–73%) the proportion of psyllids that exhibited sustainable phloem ingestion (waveform E2 > 10 min), with significant differences observed on plants treated with thiamethoxam and thiamethoxam + chlorantraniliprole. The transmission rate of CLas with high inoculum pressure (five CLas-infected D. citri per plant and a seven-day inoculation access period) to untreated control plants was 93%. In contrast, CLas transmission was reduced to 38.8% when test plants were protected by systemic insecticides. Our results indicated that all insecticides tested presented a potential to reduce CLas inoculation by an average of 59%; therefore, these insecticides can be used to reduce the spread of HLB.

Huanglongbing (HLB) is a disease of worldwide incidence that affects orange trees, among other commercial varieties, implicating in great losses to the citrus industry. The disease is transmitted through Diaphorina citri vector, which inoculates Candidatus Liberibacter spp. in the plant sap. HLB disease lead to blotchy mottle and fruit deformation, among other characteristic symptoms, which induce fruit drop and affect negatively the juice quality. Nowadays, the disease is controlled by eradication of sick, symptomatic plants, coupled with psyllid control. Polymerase chain reaction (PCR) is the technique most used to diagnose the disease; however, this methodology involves high cost and extensive sample preparation. Mass spectrometry imaging (MSI) technique is a fast and easily handled sample analysis that, in the case of Huanglongbing allows the detection of increased concentration of metabolites associated to the disease, including quinic acid, phenylalanine, nobiletin and sucrose. The metabolites abieta-8,11,13-trien-18-oic acid, suggested by global natural product social molecular networking (GNPS) analysis, and 4-acetyl-1-methylcyclohexene showed a higher distribution in symptomatic leaves and have been directly associated to HLB disease. Desorption electrospray ionization coupled to mass spectrometry imaging (DESI-MSI) allows the rapid and efficient detection of biomarkers in sweet oranges infected with Candidatus Liberibacter asiaticus and can be developed into a real-time, fast-diagnostic technique.

Made available in DSpace on 2020-12-12T02:11:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 The use of resistance inducers may be a tool to assist in the management of citrus diseases. However, knowledge about how some inducers, such as the benzothiadiazoles, promote gene expression from the pathways involved in induced resistance and alter plant development are lacking. In this work, we evaluated the effect of the acibenzolar-S-methyl (ASM) on gene expression from the biosynthetic pathways of salicylic (SA) and jasmonic (JA) acids, as well as in physiological parameters of young sweet orange trees. After a single ASM application, gene expression assessed during six time points revealed no significant changes in the main pathway for SA synthesis: isochorismate synthases (ISC) 1 and ICS2 genes were up-regulated only at 6 days after treatment (dat). However, an increase in phenylalanine ammonia lyase (PAL) was observed. Salicylic acid carboxymethyl transferase (BSMT1) and salicylic acid glucosyltransferase I (SAGT1) were up-regulated, indicating expression of genes whose products modify SA. Pointedly up-regulation of lipoxygenase (LOX) and both up- and down-regulation of allene oxide synthase (AOS) and allene oxide cyclase (AOC) in biosynthetic pathways of JA was observed, while jasmonic acid methyl transferase (JMT), whose product modify JA, was consistently induced from three to 13 dat. Besides an increase in SA and JA modification enzymes, ASM reduced photosynthetic rate in treated plants, without affecting the stomatal conductance and the transpiration rate. Our results reveal how the SA and JA pathways are regulated soon after ASM treatment, in particular the up-regulation of BSTM1, SAGT1 and JMT. Centro de Ciências Agrárias Universidade Federal de Santa Catarina - UFSC Fundo de Defesa de Citricultura - Fundecitrus, Vila Melhado Biotecnologia Instituto de Química (Programa de pós-graduação em Biotecnologia) Universidade Estadual Paulista “Júlio de Mesquita Filho” - Unesp Biotecnologia Instituto de Química (Programa de pós-graduação em Biotecnologia) Universidade Estadual Paulista “Júlio de Mesquita Filho” - Unesp

[EN] In several phytophagous hemipterans, behavior appears to be mediated by both visual and chemical cues. For the Asian citrus psyllid (ACP)Diaphorina citri(Hemiptera: Liviidae), olfactometric assays are generally difficult to interpret owing to the low proportion of individuals responding to odors (similar to 30-40%), which compromises the efficiency and reliability of the results of behavioral tests. In the present study, the ACP behavioral response to emitted odors from sweet orange (Citrus sinensisL. Osbeck) flushes in a 4-arm olfactometer using different colors (four white-, two white- and two yellow- on opposite sides, or four yellow-colored fields), and the role of the airflow in the concentration of volatile organic compounds (VOCs) were assessed at two airflows [0.4 and 0.1 L/min (LPM)]. Exposure to 'Pera' sweet orange or clean air in treatments with four yellow-colored-fields increased the response rate of ACP females to the odor sources compared with exposure to 'Pera' sweet orange or clean air in treatments with four white-colored-fields, independently of the odor source and airflow tested. For the assays using two white- and two yellow-colored fields on opposite sides and 0.4 or 0.1 LPM airflow, the residence time of ACP females to odors ('Pera' sweet orange or clean air) was similar or higher in treatments using yellow- than those using white-colored fields. For both assays (VOCs and olfactometric behavioral parameters), the reduction in airflow from 0.4 to 0.1 LPM greatly changed the airborne concentration and ACP behavior. Quantitative chemical analyses revelead that the concentration of most compounds emitted by 'Pera' sweet orange flushes for the headspace using 0.1 LPM airflow were greater than the concentrations measured using 0.4 LPM airflow. Therefore, this treatment design provides an useful tool to assess the ACP behavioral response to the odors from citrus plants, and it can also help in the discrimination of dose-response screenings for VOCs or conspecific insects., Financial support was provided by Sao Paulo Research Foundation (FAPESP) 2015/07011-3 and 2017/21460-0. The authors thank Dr. Aquidauana Miqueloto Zanardi for her help in measuring the color spaces of the white and yellow reflective papers used to change the device color.

Worldwide, there are literally hundreds of species of pathogens (fungal, bacterial, and viral) that attack citrus, yet there are only a very few that cause severe disease in epidemic proportions. Three bacterial genera contain species among these few are the subject of this chapter: (1) Xanthomonas species which requires no vector and causes hyperplastic cankers, with pathogen growth limited to intracellular mesophylic spaces of citrus leaves and stems; (2) Xylella fastidiosa subsp. pauca strains requires a vector and causes citrus variegated chlorosis (CVC), with pathogen growth limited to citrus xylem, and (3) Ca. Liberibacter species requires a vector and causes Huanglongbing (HLB), with blotchy mottling of leaves, much premature fruit drop, tree decline, and death, with pathogen growth limited to citrus phloem. Liberibacters are arguably the most destructive of all citrus pathogens, including viruses, viroids, oomycetes, bacteria, and fungi.

[EN] Production of citrus, the main fruit tree crop worldwide, is severely threatened by Huanglongbing (HLB), for which as yet a cure is not available. Spread of this bacterial disease in America and Asia is intimately connected with dispersal and feeding of the insect vector Diaphorina citri, oligophagous on rutaceous host plants. Effective control of this psyllid is an important component in successful HLB management programs. Volatiles released from the non-host guava have been shown to be repellent to the psyllid and to inhibit its response to citrus odour. By analysing VOC emission from guava we identified one volatile compound, (E)-ß-caryophyllene, which at certain doses exerts a repellent effect on D. citri. Non-host plant rejection mediated by (E)-ß-caryophyllene is demonstrated here by using Arabidopsis over-expression and knock-out lines. For the first time, results indicate that genetically engineered Arabidopsis plants with modified emission of VOCs can alter the behaviour of D. citri. This study shows that transgenic plants with an inherent ability to release (E)-ß-caryophyllene can potentially be used in new protection strategies of citrus trees against HLB., We thank Dr. Pedro Serra (IBMCP, Valencia, Spain) for his help with statistical analysis, and Prof. Dr. Luiz A.B. de Moraes (Chemistry Department, FFCLRP, USP, Riberao Preto, Brazil) and Prof. Dr. Edson Rodrigues Filho (LaBioMMi, Chemistry Department, UFSCar, Sao Carlos, Brazil) for the use of GC-MS equipment. Use of the Citrus Germplasm Bank (IVIA, Valencia, Spain) is gratefully acknowledged. This work was funded by the Fundo de Defesa da Citricultura (Fundecitrus) and FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, 2015/0711-3). In memoriam of Prof. J.M.Bove.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Citrus sudden death (CSD) transmission was studied by graft-inoculation and under natural conditions. Young sweet orange trees on Rangpur rootstock were used as indicator plants. They were examined regularly for one or two characteristic markers of CSD: (i) presence of a yellow-stained layer of thickened bark on the Rangpur rootstock, and (ii) infection with the CSD-associated marafivirus. Based on these two markers, transmission of CSD was obtained, not only when budwood for graft-inoculation was taken from symptomatic, sweet orange trees on Rangpur, but also when the budwood sources were asymptomatic sweet orange trees on Cleopatra mandarin, indicating that the latter trees are symptomless carriers of the CSD agent. For natural transmission, 80 young indicator plants were planted within a citrus plot severely affected by CSD. Individual insect-proof cages were built around 40 indicator plants, and the other 40 indicator plants remained uncaged. Only two of the 40 caged indicator plants were affected by CSD, whereas 17 uncaged indicator plants showed CSD symptoms and were infected with the marafivirus. An additional 12 uncaged indicator plants became severely affected with citrus variegated chlorosis and were removed. These results strongly suggest that under natural conditions, CSD is transmitted by an aerial vector, such as an insect, and that the cages protected the trees against infection by the vector.

Sunn hemp (Crotalaria juncea L., Fabaceae) is widely used as a cover crop in sugar cane and citrus plantations in Brazil. C. juncea has been reported in São Paulo State (SPS) by Wulff et al. (3) as a host of the phytoplasma associated with symptoms of huanglongbing (HLB) in citrus, a member of group 16SrIX, that induces witches'-broom in sunn hemp (3). In studying the distribution of group 16SrIX phytoplasma in C. juncea in SPS, we identified this species as a new host of two phytoplasmas. Sunn hemp fields were inspected for symptoms usually associated with phytoplasma infections, such as leaf yellowing, shoot proliferation, witches'-brooms, and virescence. Ninety-nine plant samples were collected and DNA was extracted with the CTAB protocol from stems. Nested PCR was carried out with primers P1/P7, followed by amplification with primers fU3/rU5 (2), both sets being universal for phytoplasma. Asymptomatic sunn hemp samples were used as negative controls and were negative in PCR reactions. PCR products were directly sequenced with primers P1/P7 and fU3/rU5 and phytoplasma identification was conducted with BLASTn and in silico RFLP analysis for delineation of subgroups (4). Plants showing leaf yellowing (three plants; Catanduva County), shoot proliferation (one plant; Ibirá County), or witches'-brooms (one plant; Promissão County) symptoms were found to be infected with the 16SrI phytoplasma group, subgroup S. The 16S rDNA sequence (GenBank Accession No. KF878383) showed 99% identity (E value 0.0) with Candidatus Phytoplasma asteris, Onion yellows phytoplasma OY-M (AP006628), Mulberry yellow dwarf phytoplasma (GQ249410), and Ash witches'-broom phytoplasma (AY566302), among other phytoplasmas from the same group. Sunn hemp plants with shoot proliferation (three plants) carried the 16SrXV phytoplasma group, subgroup A, found in Ibirá (two plants) and Catanduva (one plant) counties, SPS. This sequence (GenBank Accession No. KF878382) displayed 99% identity (E value 0.0) with Ca. P. brasiliense, Hibiscus witches'-broom phytoplasma (AF147708), Guazuma ulmifolia witches'-broom phytoplasma (HQ258882, HQ258883), and Cauliflower stunt phytoplasma (JN818845). Both phytoplasma groups described in this report, 16SrI and 16SrXV, were collected in May 2010 and both have limited geographic distribution and occurred at low incidence. Phytoplasma of group 16SrI (Ca. P. asteris) was identified in C. spectabilis in India (1). To our knowledge, this is the first report of phytoplasmas groups 16SrI and 16SrXV in sunn hemp. References: (1) S. Kumar et al. Plant Dis. 94:1265, 2010. (2) E. Seemüller et al. Int. J. Syst. Bacteriol. 44:440, 1994. (3) N. A. Wulff et al. Tropical Plant Pathol. 34:S7, 2009. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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Huanglongbing (HLB) is a citrus disease of worldwide importance, associated with the presence of Candidatus Liberibacter asiaticus (Las) and vectored by the psyllid Diaphorina citri in Asia and the Americas. To properly manage HLB, removal of inoculum sources and control of the psyllid are undertaken. We evaluated the percentage of the psyllid population with Las, sampled from yellow sticky traps over a three-year period and its relationship with insect population, regions, season of the year, and HLB management in citrus areas in the southwestern, central, and northern regions of S&atilde, o Paulo (SP) and southwestern region of Minas Gerais states, Brazil. In each reading, up to 50 psyllids per region were collected and detection of Las in individual psyllids were made by quantitative polymerase chain reaction. The percentage of psyllids with Las&mdash, an average of 65.3%&mdash, was constant throughout the year in the southwestern region of SP state, while showing an increase from spring to autumn when sampled from central to northern regions. The proportion of psyllids carrying Las from each region and year period were compared by a proportion test and spectral density analysis. The proportion of psyllids carrying Las evaluated in the same region in different seasons presented statistical differences in central (Araraquara) and southwestern (Santa Cruz do Rio Pardo) regions in 2015, with higher values in the first semester (summer and autumn) than in the second semester (winter and spring). Orchards with poor HLB management had higher incidence of psyllids with Las. Spectral density analysis indicated that good management areas had 50% less relevant peaks of psyllids with Las than in areas with poor HLB management practices. The relationship between the percentage of psyllids carrying Las and the number of captured psyllids in the region in a given time denotes the most critical intake time for HLB spread in citrus orchards. The reduction in the population of psyllids carrying Las is a direct benefit from the use of good management practices.

UMR BFP - Equipe Mollicutes; International audience; When huanglongbing (HLB) was found in Brazil in 2004, 'Candidatus Liberibacter americanus' was infecting most of the trees while 'Ca. L. asiaticus' was present in a minor proportion. Currently, 'Ca. L. asiaticus' is the predominant bacterium associated with HLB in citrus trees in São Paulo (SP) and Minas Gerais (MG) States, the major citrus-growing regions in Brazil. A phytoplasma from the 16SrIX group was associated with HLB symptoms in Brazil in 2007, in plants free of Liberibacter spp. In this report, HLB samples testing negative for 'Ca. L. asiaticus', 'Ca. L. americanus', and 16SrIX phytoplasma were infected with 16SrIII phytoplasmas. Coinfection with 'Ca. L. asiaticus' and 16SrIII was also found. The 16S ribosomal RNA (rRNA) gene sequences from 22 samples were obtained and sequenced, confirming that the 16SrIII group phytoplasma is associated with HLB symptoms in SP and MG States. Ten single-nucleotide polymorphisms (SNPs) were found in the 1,427-bp 16S rRNA gene sequences from 16SrIII phytoplasmas from citrus, whereas none was detected in 16S rRNA gene sequences among 16SrIX phytoplasma from citrus. Ribosomal protein (rp) rpsSrplVrpsC gene sequences were amplified with 16SrIII group-specific primers, sequenced from a subset of nine samples, and assembled into three groups based on eight SNPs. SNPs in 16S rRNA gene and rp gene sequences are common in 16SrIII phytoplasmas from other hosts and this phytoplasma group is widespread in South America. 16SrIII phytoplasmas highly related are commonly found in Melia azedarach, a widespread tree in Brazil and Argentina. The finding of a new phytoplasma associated with HLB symptoms belonging to the 16SrIII group reinforces the need to develop diagnostic tools to assess HLB-associated microbiomes.

Citrus sudden death (CSD) is a highly destructive disease and has caused the eradication of millions of trees in southern Brazil within the last 15 years. In spite of the exact cause of CSD has not been determined, evidences have shown that this disease can be transmitted by biotic vectors. Disease incidence in sweet orange scions is related to the rootstock, and the combination with ‘Rangpur’ lime is the most affected. On the other hand, there are evidences of a relation between CSD affected trees and the presence of the Citrus sudden death associated virus (CSDaV) and/or Citrus tristeza virus (CTV). Based on such information, this study has been carried out to determine the presence of CSDaV and CTV, and the association between each other in eleven rootstocks for ‘Valencia’ sweet orange. The results presented herein showed differences related to the presence of CSDaV and CTV in different rootstocks for ‘Valencia’ sweet orange and no relation between the presence of CSDaV and CTV. Resumo A morte súbita dos citros (MSC) é uma doença de alto impacto destrutivo e tem levado à erradicação de milhões de plantas de laranja-doce na região Sudeste do Brasil, nos últimos 15 anos. Embora o agente causal da MSC ainda não tenha sido determinado, evidências demonstraram que esta doença pode ser transmitida por um vetor biótico. A incidência de MSC em cultivares de laranja-doce está relacionada ao porta-enxerto, sendo que a combinação com limoeiro ‘Cravo’ é a mais afetada. Por outro lado, há evidências de relação entre plantas sintomáticas para MSC e a presença de um vírus, denominado Citrus sudden death associated virus (CSDaV) e/ou com Citrus tristeza virus (CTV). Com base nessas informações, o objetivo deste estudo foi determinar a presença de CSDaV e CTV e a associação entre eles, em onze porta-enxertos para laranjeira ‘Valencia’. Os resultados apresentados aqui sugerem diferenças relacionadas à presença de CSDaV e CTV em diferentes porta-enxertos para laranja ‘Valencia’, não havendo relação entre a presença de CSDaV e CTV.

Two surveys (2005 ⁄ 2006 and 2009) were conducted in the state of Sao Paulo, Brazil, to investigate the incidence of 'Candid- atus Liberibacter asiaticus' and 'Ca. L. americanus', two liberibacters associated with citrus huanglongbing (HLB) disease and both transmitted byDiaphorina citri ,i n orange jasmine ( Murraya exotica), a widespread ornamental tree in cities and villages. The graft-transmissibility of the two species, and their DNA relatedness to citrus-associated liberibacters, were also investigated. Quantitative PCR was applied to PCR-positive orange jasmine and HLB-positive citrus growing in backyards and orchards to assess their inoculum source potentials. Liberibacters were detected in 91 of 786 sampled orange jasmine plants in 10 of 76 sampled locations. PCR-positive trees exhibited yellow shoots and ⁄ or dieback symptoms indistinguishable from those on PCR-negative trees. 'Candidatus Liberibacter americanus' was more common in 2005 ⁄ 2006 (96AE6%) and 'Ca. L. asiaticus' in 2009 (84AE8%). rplJ nucleotide sequences were identical within all populations of either species. Graft transmission succeeded only in homologous host combinations, including 'Ca. L. americanus' (2 ⁄ 10) from ⁄ to orange jas- mine and 'Ca. L. americanus' (5 ⁄ 18) and 'Ca. L. asiaticus' (5 ⁄ 9) from ⁄ to citrus. Symptoms were mild and developed less rapidly in orange jasmine than in citrus, probably as a result of lower liberibacter multiplication rates. Respective titres of 'Ca. L. americanus' and 'Ca. L. asiaticus' in orange jasmine averaged 4AE 3a nd 3AE0 log cells g )1 tissue, compared with 5AE 5a nd 7AE3 in citrus. The results indicate that orange jasmine does not favour liberibacter multiplication as much as citrus. However, its importance in HLB epidemics should not be underestimated as it is a preferred host of D. citri and is not under any strict tree-eradication programme or measures for insect control.

Os recentes relatos da presenca do huanglongbing (HLB) em algumas das maiores regioes produtoras de citros em paises das Americas do Sul, Central e do Norte, principalmente o estado de Sao Paulo no Brasil e o estado da Florida nos Estados Unidos, confirmam a grande capacidade de disseminacao e progresso desta devastadora doenca que coloca em risco a sustentabilidade do agronegocio citricola nestes paises. Anteriormente a estes relatos, poucos estudos epidemiologicos quantitativos haviam sido conduzidos por causa das dificuldades e dos riscos de se manter estes estudos por varios anos seguidos em pomares sem nenhum controle e pela propria natureza complexa deste patossistema, que inclui um longo periodo de incubacao e sazonalidade de expressao dos sintomas. Apesar disso, nos ultimos anos, informacoes uteis tem sido obtidas por estudos epidemiologicos realizados em Sao Paulo e na Florida com respeito aos processos espaco-temporais que levam ao aumento do HLB, isto e, como a doenca se dissemina e como progride. Estas informacoes sao uteis para o estabelecimento e validacao de estrategias eficientes de manejo desta doenca, assim como para estimar a longevidade economica e biologica de um determinado pomar sob determinadas condicoes de manejo. O objetivo desta revisao e fornecer informacoes atualizadas sobre a caracterizacao das epidemias de HLB e alertar os citricultores e agentes de defesa fitossanitaria sobre a importância da adocao imediata de medidas quarentenarias para impedir a introducao do HLB em areas livres da doenca, assim como da importância da adocao de estrategias regionais de controle.

International audience; Huanglongbing is one of the most severe diseases of citrus worldwide and is associated with ‘Candidatus (Ca.) Liberibacter africanus’ in Africa, ‘Ca. Liberibacter asiaticus’ in Asia and the Americas (Brazil, USA and Cuba) and ‘Ca. Liberibacter americanus’ (Lam) in Brazil. In the absence of axenic cultures, genetic information on liberibacters is scarce. The sequences of the entire 23S rRNA and 5S rRNA genes from Lam have now been obtained, using a consensus primer designed on known tRNAMet sequences of rhizobia. The size of the Lam genome was determined by PFGE, using Lam-infected periwinkle plants for bacterial enrichment, and was found to be close to 1.31 Mbp. In order to determine the number of ribosomal operons on the Lam genome, probes designed to detect the 16S rRNA gene and the 3' end of the 23S rRNA gene were developed and used for Southern hybridization with I-CeuI-treated genomic DNA. Our results suggest that there are three ribosomal operons in a circular genome. Lam is the first liberibacter species for which such data are available.

Citrus sudden death (CSD) is a new disease of sweet orange and mandarin trees grafted on Rangpur lime and Citrus volkameriana rootstocks. It was first seen in Brazil in 1999, and has since been detected in more than four million trees. The CSD causal agent is unknown and the current hypothesis involves a virus similar to Citrus tristeza virus or a new virus named Citrus sudden death‐associated virus. CSD symptoms include generalized foliar discoloration, defoliation and root death, and, in most cases, it can cause tree death. One of the unique characteristics of CSD disease is the presence of a yellow stain in the rootstock bark near the bud union. This region also undergoes profound anatomical changes. In this study, we analyse the metabolic disorder caused by CSD in the bark of sweet orange grafted on Rangpur lime by nuclear magnetic resonance (NMR) spectroscopy and imaging. The imaging results show the presence of a large amount of non‐functional phloem in the rootstock bark of affected plants. The spectroscopic analysis shows a high content of triacylglyceride and sucrose, which may be related to phloem blockage close to the bud union. We also propose that, without knowing the causal CSD agent, the determination of oil content in rootstock bark by low‐resolution NMR can be used as a complementary method for CSD diagnosis, screening about 300 samples per hour.

The major feature of Xylella fastidiosa growing in its hosts, as well as in culture media, is its cellular aggregation and biofilm formation, leading to partial obstruction of the xylem causing water stress in the plant. We report that growth, aggregation, and biofilm formation of X. fastidiosa are influenced by the medium pH. We have verified that X. fastidiosa cell aggregation is reversibly inhibited by decreasing the medium pH from 6.6 to 6.4. Biofilm formation on glass walls was affected as well, and a concomitant decrease in cell multiplication was observed below pH 6.4. The manipulation of culture medium pH can be used as a tool for the cloning of X. fastidiosa strains isolated from plant hosts, because different strains can inhabit the same plant. Also, X. fastidiosa mutants produced by gene manipulation can be isolated from cell aggregates containing transformed and untransformed cells.

This paper introduces the basics of peptide mass spectra interpretation applied to proteomics and is directed to chemists, biochemists and biologists. The manuscript presents a well detailed protocol aiming to serve as a first choice guide for understanding peptide sequencing. The tutorial was elaborated based on both a thorough bibliographic revision and the author's experience. In order to prove the applicability of the proposed guide, spectra obtained on different instruments have been successfully interpreted by applying the presented rational.

A bactéria Xylella fastidiosa causa a clorose variegada dos citros, também conhecida como amarelinho. Em 2000, foi concluído o seqüenciamento do genoma deste organismo, o primeiro de uma bactéria fitopatogênica, um fato que estimulou o estudo dos possíveis mecanismos de patogenicidade empregados pela bactéria para causar a doença em citros. X. fastidiosa é uma bactéria limitada ao xilema, sendo transmitida de planta a planta através de insetos vetores. Especula-se que a bactéria produza enzimas que degradem a membrana da pontuação, permitindo a colonização de novos vasos do xilema. A identificação de genes com similaridade de seqüência a genes de celulases, xilanases, pectinases e proteases, fomentou o presente estudo visando caracterizar os genes Xf - 810, Xf - 818 e Xf - 2708, similares a endoglicanases. Estes genes foram clonados em vetores de expressão e as respectivas proteínas foram produzidas em Escherichia coli. Através de ensaios enzimáticos as proteínas foram caracterizadas como endoglicanases (EC 3.2.1.4), que são celulases com mecanismo endoglicolítico de ataque às moléculas de celulose. Estas celulases hidrolisam carboximetil celulose, Avicel e xilana, enquanto as enzimas Xf - 810 e Xf - 818 hidrolisam a celulose amolecida com ácido. Estas celulases degradam mais eficientemente a carboximetil celulose em pH ácido (entre 5,2 e 5,6) e na temperatura de 65 °C. Coletivamente, estas celulases são capazes de degradar polímeros solúveis e insolúveis, enquanto a enzima Xf - 818, é capaz de degradar oligossacarídeos derivados da celulose, como celotetraose e celopentaose, apresentando ampla variação catalítica. Esta celulase possui capacidade de ligação à celulose microcristalina, denotando a funcionalidade de seu domínio ligador de celulose. Desenvolvemos um protocolo, empregando cromatografia de troca aniônica, afinidade por metal (níquel) e filtração em gel, eficiente na purificação da proteína Xf - 818 expressa heterologamente em E. coli com fusão hexahistidina à extremidade N-terminal. A caracterização enzimática destas proteínas, com a confirmação da atividade celulásica, fornece subsídios para uma eventual função das celulases durante a colonização do hospedeiro pela bactéria, pois são cataliticamente funcionais. Ademais, corrobora a similaridade destes genes, verificada durante o seqüenciamento do genoma de X. fastidiosa. Xylella fastidiosa is the causal agent of citrus variegated chlorosis, also known as "amarelinho". The recent sequencing of its genome, achieved in 2000, was the first of a plant pathogen, a fact that stimulated the search for putative pathogenicity factors employed by this bacterium while infecting citrus trees. X. fastidiosa inhabits exclusively the xylem vessels, being transmitted by sharpshooter vectors. Several authors argue that the bacterium produces enzymes to degrade plant cell, as a way to colonize new xylem vessels through pit membrane degradation. The identification of putative cellulases, xylanases, pectinases and proteases on X. fastidiosa genome, led us to carry out the present work to characterize the putative products of the endoglucanase genes Xf - 810, Xf - 818 and Xf - 2708. These genes were cloned into expression vectors and the proteins were produced in Escherichia coli. Based upon enzymatic assays, those proteins were characterized as endoglucanases (EC 3.2.1.4), which are cellulases able to promote the endo-hydrolysis of cellulose chains. These cellulases degraded carboxymethylcellulose, Avicel and xylan, while only Xf - 810 and Xf - 818 degraded acid swollen cellulose. The hydrolysis of carboxymethylcellulose was higher at acidic pH between 5.2 and 5.6) and at a temperature of 65 °C. As a group, these enzymes were able to degrade soluble and insoluble cellulose derivatives, while only the cellulase Xf - 818 could hydrolyze the cello-oligosaccharides cellotetrose and cellopentose, thus showing a high catalytic diversity. This protein also has the capacity to bind microcristalline cellulose, confirming the presence of a functional cellulose-binding domain. We set a protocol, employing anion exchange, metal affinity and gel filtration chromatography, to purify the Xf - 818 enzyme expressed in E. coli as a N-hexahistine fusion tag. The endoglucanase activity studied gives support for an eventual role of such cellulases during host colonization by the bacterium. Besides that, it agrees with similarities searches observed on the sequencing of X. fastidiosa genome.

In São Paulo State (SPS), sweet orange (Citrus sinensis) trees with huanglongbing (HLB) symptoms are infected with Candidatus (Ca.) Liberibacter (L.) asiaticus (Las) or Ca. L. americanus (Lam). However, in 2007, 3 years after HLB was first reported in SPS, some trees with characteristic HLB symptoms were found free of liberibacters, but infected with a phytoplasma of 16Sr group IX. This phytoplasma was further characterized by PCR amplification of ribosomal protein genes rpsC-rplV-rpsS and amplicon sequencing. A qPCR test to detect the phytoplasma in plants and insects was also developed on the basis of the ribosomal protein genes. The phytoplasma was transmitted from citrus-to-citrus by grafting. The 16Sr group IX phytoplasma associated with HLB symptoms in sweet orange in SPS and characterized by the above techniques was named “HLB-phytoplasma”. Although the HLB-phytoplasma is widely distributed in many municipalities of central, northern, and northwestern SPS, the number of HLB-phytoplasma-infected trees in each municipality is very small. Experiments have been undertaken to identify the origin of the HLB-phytoplasma and the source of inoculum on which a putative insect vector could become infected with the HLB-phytoplasma. In SPS, sunn hemp (Crotalaria juncea L.) is a major, widely distributed cover crop. A 16Sr group IX phytoplasma was detected in sunn hemp plants with witches’ broom and virescence symptoms, and was shown to have 16Sr DNA sequences and ribosomal protein gene sequences with 100% identity to the corresponding sequences of the sweet orange HLB-phytoplasma. Transmission electron microscopy revealed the presence of phytoplasma cells in the phloem sieve tubes of infected C. juncea stalks. These results were taken as evidence that the sunn hemp phytoplasma and the sweet orange HLB-phytoplasma were identical. Scaphytopius marginelineatus, a leafhopper frequently found in sweet orange orchards, was shown to acquire the HLB-phytoplasma efficiently from affected sunn hemp plants, but acquisition from, and transmission rates to, sweet orange were very low. On the whole, these data suggest that (i) sunn hemp is a major source of inoculum of the HLB-phytoplasma, (ii) S. marginelineatus becomes infected on sunn hemp and transmits the phytoplasma to sweet orange, and (iii) transmission from sweet orange to sweet orange occurs only rarely, if at all. 16Sr group IX phytoplasmas, very closely related to the SPS HLB-phytoplasma, have also been detected in citrus in Minas Gerais and Bahia states (Brazil) and Mexico.

Xylella fastidiosa was the first plant pathogen whose complete genome sequence was available. X. fastidiosa causes citrus variegated chlorosis, but the physiological basis of the disease in unknown. Through comparative sequence analysis, several putative plant cell wall-degrading enzymes were identified on the X. fastidiosa genome. We have cloned Xf818, a putative endoglucanase ORF, into expression vectors pET20b and pET28b, and purified a recombinant form of Xf818 containing a His(6) tag. Through biochemical assays, we have characterized the endoglucanase activity of this protein. The best conditions for hydrolysis over carboxymethyl cellulose (CMC) were on pH 5.2 at 65 degrees C. Xf818 hydrolyzed CMC, acid swollen cellulose, Avicel, birch wood, oat spels xylans, and the oligosaccharides cellotetraose and cellopentaose. Xf818 carried out transglycosylation and had a functional cellulose-binding domain.

The shiitake mushroom (Lentinula edodes) has been used in research involving the prevention and the control of human and plant diseases. In cucumber plants, treated with aqueous extracts from mushroom basidiocarps, there was a reduction in anthracnose severity caused by Colletotrichum lagenarium, and an increase in peroxidase activity in the leaves. With the aim of obtaining molecules of agronomic interest, the crude aqueous extract from L. edodes basidiocarp was fractioned with ammonium sulfate. The fraction corresponding to 40-80% of saturation (p40-80), the most effective in reducing anthracnose on cucumber cotyledons, was submitted to anion exchange chromatography (AEC). After AEC, six protein peaks were obtained and the peak V, containing 34% of the proteins present in p40-80, induced peroxidase increase in the cucumber cotyledons besides reducing anthracnose severity. Separation of peak V proteins by SDS-gel electrophoresis revealed the presence of more than one band in the gel. Thus, a partial purification of elicitors present in the L. edodes basidiocarp was achieved. O cogumelo shiitake (Lentinula edodes) vem sendo utilizado com objetivos de prevenir e controlar doenças em humanos e em vegetais. O tratamento com o extrato aquoso de basidiocarpos do cogumelo propiciou uma redução significativa na severidade da antracnose em plantas de pepino, provocada por Colletotrichum lagenarium, e um aumento na atividade de peroxidases nos tecidos foliares. Desse modo, com o objetivo de obter moléculas de interesse agronômico destinadas à proteção de plantas, o extrato aquoso bruto de basidiocarpos de L. edodes foi fracionado inicialmente com sulfato de amônio. A fração correspondente a 40-80% de saturação (p40-80) foi a mais efetiva em reduzir a antracnose em cotilédones de pepino, sendo então submetida à cromatografia de troca aniônica (CTA). Após a CTA, seis picos protéicos foram obtidos, e o pico V, contendo em média 34% das proteínas presentes em p40-80, provocou o acúmulo de peroxidases nos cotilédones de pepino, bem como reduziu a antracnose. A separação eletroforética das proteínas do pico V revelou a presença de mais de uma banda no gel de poliacrilamida. Portanto, estes procedimentos resultaram na purificação parcial de elicitores presentes no basidiocarpo de L. edodes.

In São Paulo State, besides the occurrence of Candidatus Liberibacter americanus and Ca. L. asiaticus, a 16Sr IX group phytoplasma was associated with HLB symptoms, indistinguishable from those caused by liberibacters. This phytoplasma is called HLB phytoplasma and was found widespread in citrus orchards, although at low incidence. The same phytoplasma was found in Sunn Hemp (Crotalaria juncea) in 2008 and witches’-broom was commonly found associated with 16Sr group IX detection. The aim of this work was to assess the phytoplasma diversity in Sunn Hemp with emphasis at the detection of group 16Sr IX phytoplasma and to establish an association between the occurrence of HLB phytoplasma and symptoms. Sunn Hemp samples were harvested close to the blooming period. Plants were selected in the field when showing symptoms common to phytoplasma infection. We employed universal primers do amplify phytoplasmas in general and group specific primers for 16Sr group IX. PCR products were sequenced to allow grouping of phytoplasmas. We identified five phytoplasmas groups in 48 out of 99 Sunn Hemp plants, belonging to phytoplasma groups 16Sr I, III, VII, IX and XV. The most abundant phytoplasma was the group 16Sr IX, present in 70% of the samples, found in central and north São Paulo State. The occurrence of HLB phytoplasma in Sunn Hemp samples, showing 100% of similarity to the citrus phytoplasma, was highly related to virescence and the second most conspicuous symptom for this infection was witches’-broom.

We used PFGE followed by CsCl bisbenzymide centrifugation to obtain sufficient DNA for pyrosequencing of the Ca. Liberibacter americanus (Lam) strain “Sao Paulo” genome. The complete circular genomic DNA sequence of Lam is 1,195,201 bp, with an average GC content of 31.12%, somewhat lower than other Liberibacters. There are 1,056 predicted Lam genes, with 1,002 encoding proteins, 9 encoding rRNA genes and 45 encoding tRNAs. The overall gene organization and structure of the Lam genome is more similar to Lso than to Las. There are 951 genes common to Lam, Lso and Las, 27 genes found in Lam and Lso but not Las, and only 8 genes common to Lam and Las but not found in Lso. Many pseudogenes or truncated genes were found among the unique genes of all 3 species. As with Las, two prophage were confirmed in Lam, with SP2 being 39,941 bp and SP1 being 16,398 bp in size; as in Las, the one that appears to replicate as an excision plasmid prophage carries putative lysogenic conversion genes, specifically peroxidases and a Type Vc secreted adhesin. These predicted peroxidases and adhesin were found in both Las and Lam, but appeared fragmentary or degenerated in Lso, indicating their potential for citrus host range determination. Liberibacter genomes seem to be under selective pressure to reduce GC% content and to lose unimportant genes. Although Lam have an outer membrane, most of the genes required for biosynthesis of lipopolysaccharide, which can trigger ROS production, are missing from the Lam genome.

Author(s): Zhang, S. J.; Wulff, N. A.; Fleites, L. A.; Zhang, Y. C.; Gabriel, D. W. | Abstract: Huanglongbing (HLB) is a lethal disease of citrus caused by Ca. L. asiaticus (Las), Ca. L. americanus (Lam), and Ca. L. africanus. Our published results demonstrate that Las carries a prophage with a lytic cycle that can become activated in plants to kill the Las cell that carries it. Our more recent results analyzing the complete genome of Lam (refer Wulff et al abstract at this conference) demonstrates that it, too, carries a very similar prophage and apparent lytic cycle. Our goal is to try to develop a sensitive, multiwell, microtiter dish assay for high throughput screening of chemicals with ability to trigger the lytic cycle and potentially lead to a chemical treatment method to eliminate Las from infected trees, whether symptomatic with HLB or not. The intergenic region between the early and late genes of Las phage SC1 and SC2 (between locus tags gp120 and gp125) were cloned in both directions upstream of the lacZ reporter gene in E. coli. We then cloned and expressed predicted repressors and anti-repressors from Las to determine responsiveness of the reporter constructs. As expected, the predicted early gene reporters of both SC1 and SC2 were constitutively on and the late genes were constitutively off. However, the predicted repressors and antirepressors failed to function as predicted and a detailed examination of the intergenic region revealed that late gene expression is likely initiated in at least one other location. Additional putative late gene promoter regions are being analyzed.

The assessment of bacterialiferous Asian citrus psyllid (ACP) frequency is important for (i) studies of bacteria acquisition and inoculation by ACP, (ii) disease detection in disease free areas but with ACP presence, (iii) efficiency evaluation of inoculum reduction strategies, (iv) evaluation of frequency of Candidatus Liberibacter asiaticus (Las)-positive ACP and the abundance of inoculum sources or putative new HLB infections relationships. Depending on the conditions and time of storage of collected psyllids, Las DNA in ACP could degrade and Las-false negative results might occur. Thus, this study was conducted to evaluate the detection of Las in ACP adults submitted to different storage methods and time of storage by real-time PCR (qPCR). Two 2x3x7 factorial experiments were conducted. Factors were ‘Ethanol’ (with or without 70% ethanol), ‘Temperature’ (-20°C, 4°C and 26°C) and ‘Time’ (0, 3, 7, 14, 21, 28 and 35 days). For each treatment, 20 samples with 3 ACP adults from nymphs reared on Las infected trees were tested for Las presence by qPCR. No significant differences in percentages of psyllids samples positive for Las were observed among the storage methods up to 35 days, except a slight trend of decline in Las detection in samples storage without ethanol at 26°C after 14 days of storage.