Alfalfa leaf curl virus is transmitted by Aphis craccivora in a highly specific circulative manner.

Two members of the genus Capulavirus (Geminiviridae) are transmitted by aphids including Alfalfa leaf curl virus (ALCV) transmitted by Aphis craccivora. The capulavirus Euphorbia caput-medusae latent virus was shown here to be transmitted also by A. craccivora, using the population EuphorbiaSA. ALCV was transmissible by several A. craccivora populations including Robinia, but not the EuphorbiaSA population, reflecting a high transmission specificity. Typical of the circulative-persistent mode of transmission, ALCV persists through insect molts. ALCV accumulation and localization were analyzed in whole insects, midguts, hemolymphs, and heads of aphids from vector and non-vector populations of A. craccivora and from the non-vector species Acyrthosiphon pisum. Vector and non-vector populations could be distinguished by contrasted virus accumulations and midgut intracellular localization consistent with a gut barrier to the transmission of ALCV in A. pisum and a primary salivary gland barrier in A. craccivora.

Viral Metagenomic-Based Screening of Sugarcane from Florida Reveals Occurrence of Six Sugarcane-Infecting Viruses and High Prevalence of Sugarcane yellow leaf virus.

A viral metagenomics study of the sugarcane virome in Florida was carried out in 2013 to 2014 to analyze occurrence of known and potentially new viruses. In total, 214 sugarcane leaf samples were collected from different commercial sugarcane (Saccharum interspecific hybrids) fields in Florida and from other Saccharum and related species taken from two local germplasm collections. Virion-associated nucleic acids (VANA) metagenomics was used for detection and identification of viruses present within the collected leaf samples. VANA sequence reads were obtained for 204 leaf samples and all four previously reported sugarcane viruses occurring in Florida were detected: Sugarcane yellow leaf virus (SCYLV, 150 infected samples out of 204), Sugarcane mosaic virus (1 of 204), Sugarcane mild mosaic virus (13 of 204), and Sugarcane bacilliform virus (54 of 204). High prevalence of SCYLV in Florida commercial fields and germplasm collections was confirmed by reverse-transcription polymerase chain reaction. Sequence analyses revealed the presence of SCYLV isolates belonging to two different phylogenetic clades (I and II), including a new genotype of this virus. This viral metagenomics approach also resulted in the detection of a new sugarcane-infecting mastrevirus (recently described and named Sugarcane striate virus), and two potential new viruses in the genera Chrysovirus and Umbravirus.

Increase in taxonomic assignment efficiency of viral reads in metagenomic studies.

Metagenomics studies have revolutionized the field of biology by revealing the presence of many previously unisolated and uncultured micro-organisms. However, one of the main problems encountered in metagenomic studies is the high percentage of sequences that cannot be assigned taxonomically using commonly used similarity-based approaches (e.g. BLAST or HMM). These unassigned sequences are allegorically called « dark matter ¼ in the metagenomic literature and are often referred to as being derived from new or unknown organisms. Here, based on published and original metagenomic datasets coming from virus-like particle enriched samples, we present and quantify the improvement of viral taxonomic assignment that is achievable with a new similarity-based approach. Indeed, prior to any use of similarity based taxonomic assignment methods, we propose assembling contigs from short reads as is currently routinely done in metagenomic studies, but then to further map unassembled reads to the assembled contigs. This additional mapping step increases significantly the proportions of taxonomically assignable sequence reads from a variety -plant, insect and environmental (estuary, lakes, soil, feces) - of virome studies.

Discovery of parvovirus-related sequences in an unexpected broad range of animals.

Our knowledge of the genetic diversity and host ranges of viruses is fragmentary. This is particularly true for the Parvoviridae family. Genetic diversity studies of single stranded DNA viruses within this family have been largely focused on arthropod- and vertebrate-infecting species that cause diseases of humans and our domesticated animals: a focus that has biased our perception of parvovirus diversity. While metagenomics approaches could help rectify this bias, so too could transcriptomics studies. Large amounts of transcriptomic data are available for a diverse array of animal species and whenever this data has inadvertently been gathered from virus-infected individuals, it could contain detectable viral transcripts. We therefore performed a systematic search for parvovirus-related sequences (PRSs) within publicly available transcript, genome and protein databases and eleven new transcriptome datasets. This revealed 463 PRSs in the transcript databases of 118 animals. At least 41 of these PRSs are likely integrated within animal genomes in that they were also found within genomic sequence databases. Besides illuminating the ubiquity of parvoviruses, the number of parvoviral sequences discovered within public databases revealed numerous previously unknown parvovirus-host combinations; particularly in invertebrates. Our findings suggest that the host-ranges of extant parvoviruses might span the entire animal kingdom.

A Novel Itera-Like Densovirus Isolated by Viral Metagenomics from the Sea Barley Hordeum marinum.

Densoviruses (DVs) infect arthropods and belong to the Parvoviridae family. Here, we report the complete coding sequence of a novel DV isolated from the plant Hordeum marinum (Poaceae) by viral metagenomics, and we confirmed reamplification by PCR. Phylogenetic analyses showed that this novel DV is related to the genus Iteradensovirus.

First Report of Acidovorax avenae subsp. avenae Causing Sugarcane Red Stripe in Gabon.

During a disease inspection at the sugarcane estate SUCAF near Franceville, Gabon, in March 2011, 1- to 3-mm wide and several dm long dark red stripes were observed on sugarcane (Saccharum spp.) leaves of many plants of cultivar R570. These plants were 5.5 months old in the first ratoon crop. Additionally, spindle leaves of several stalks were rotted and could be easily pulled out of the top of the stalk. Longitudinal sections of diseased stalks showed reddish-brown discoloration of the upper stem and the rotted spindle had an unpleasant odor. Circular, convex, smooth, yellow-cream pigmented bacterial colonies with 2 to 3 mm diameter were isolated after 3 days at 28°C from young leaf lesions on YPGA (yeast extract 7 g/L, peptone 7 g/L, glucose 7 g/L, agar 15 g/L, pH 6.8 to 7.0). The 16S-23S internal transcribed spacer (ITS) of two representative colonies was PCR amplified, and the nucleotide sequences were shown to be 99% identical to the 16S-23S ITS sequence from the genome of Acidovorax avenae subsp. avenae strain ATCC 19860 (GenBank: CP002521.1). One of these A. avenae subsp. avenae isolates from Gabon was inoculated into greenhouse grown plants of sugarcane cultivar R570. Plants were inoculated by injection into the sheath of spindle leaves above the meristem with the bacterial strain (12 plants) or with a water control (six plants). In this method, the bacteria (108 CFU/ml) were injected using a syringe through the leaf sheath until filling the leaf whorl. Three weeks post-inoculation, one to several cm long red-brown stripes were observed on leaves of 11 of 12 inoculated plants. Seven weeks post-inoculation, all plants exhibited symptoms, from red, brown, or black stripes to leaf necrosis, rotting, and death of the spindle leaves (six plants). All six control plants were symptomless. In a second experiment, 6 of 12 plants showed symptoms 3 weeks post inoculation, and the pathogen was successfully re-isolated from all six symptomatic plants with YPGA medium. The 16S-23S ITS of three single colonies obtained each from different symptomatic plants were PCR amplified and the nucleotide sequences were again found 99% identical to the 16S-23S ITS sequence from the genome of A. avenae subsp. avenae ATCC 19860. To our knowledge, this is the first report of A. avenae subsp. avenae, the causal agent of sugarcane red stripe (also reported as top rot), in Gabon. It is also the first description of the occurrence of the top rot form of the disease in R570, a cultivar that is grown in several locations of Africa, the Mascarene Islands, and the French West Indies. A large-scale survey needs to be undertaken to determine the distribution of red stripe in Gabon, a disease for which several outbreaks have been reported recently worldwide (1,2). References: (1) M. P. Grisham and R. M. Johnson. Phytopathology 101:564, 2011. (2) S. Zia-ul-Hussnain et al. Afr. J. Biotechnol. 10:7191, 2011.

First Report of Sugarcane Leaf Scald in Gabon Caused by a Highly Virulent and Aggressive Strain of Xanthomonas albilineans.

During a disease inspection at the sugarcane estate SUCAF near Franceville, Gabon, in March 2011, 0.5 to 1 cm wide chlorotic stripes covered with many small red streaks were observed on sugarcane (Saccharum spp.) leaves of a single plant of cultivar R581. After removal of the leaves covering the base of the stalks, abnormal development of basal side shoots was also observed. Transversal sections of a diseased stalk showed reddening of the vessels near the nodes. Circular, convex, smooth, shiny, translucent, non-mucoid, honey-yellow pigmented bacterial colonies were isolated from stalk pieces and side shoots on XAS selective agar medium (1). The nucleotide sequence of the 16S-23S internal transcribed spacer (ITS) of a representative colony was shown to be 100% identical to the 16S-23S ITS sequence from the genome of Xanthomonas albilineans strain GPE PC73 (GenBank: FP565176.1). This strain from Gabon was named GAB266. Sugarcane stalks of greenhouse grown cultivar CP68-1026 were inoculated with X. albilineans strains XaFL07-1 from Florida, GPE PC73 from Guadeloupe, and GAB266. Five stalks were inoculated by the modified decapitation method (3) with each strain or with a water control. One month post-inoculation (MPI), white pencil lines and severe necrosis were observed on leaves inoculated with strains XaFL07-1 and GPE PC73, and no disease symptoms appeared on non-inoculated leaves that developed 2 to 3 MPI. These results are in agreement with those generally obtained after inoculation of susceptible sugarcane cultivars with X. albilineans strains from various geographical locations under greenhouse conditions (Rott, unpublished results). In contrast, 1 MPI, only discrete white to red pencil lines were observed along with necrosis on leaves inoculated with strain GAB266, and by 2 to 3 MPI, all five inoculated plants were wilted. The pathogen was successfully reisolated by the stalk blot inoculation technique (3) with XAS medium, from all five inoculated stalks and from 98 of 114 internodes. In a second greenhouse experiment, the same three strains of X. albilineans were inoculated as described above into five sugarcane cultivars differing in resistance to leaf scald in Guadeloupe (2) (CP68-1026, highly susceptible; B69566, susceptible; R570, tolerant; B8008, resistant; Co6415, highly resistant). The same symptoms as those described above were again observed on inoculated leaves of the five sugarcane cultivars 1 MPI. Strains XaFL07-1 and GPE PC73 produced occasionally a single pencil line on non-inoculated leaves 2 to 3 MPI, but only strain GAB266 caused leaf scalding and/or plant death 2 to 3 MPI: cultivar CP68-1026 (5 of 5 plants), B69566 (5 of 5 plants), R570 (4 of 5 plants), B8008 (5 of 5 plants), and only non-inoculated leaves of cultivar Co6415 remained symptomless (5 plants). Strain GAB266 from Gabon appeared, therefore, more virulent and aggressive than the two strains of X. albilineans from Florida and Guadeloupe. To our knowledge, this is the first report of leaf scald of sugarcane in Gabon and the first description of an unusual highly virulent and aggressive strain of X. albilineans. A large-scale survey needs to be undertaken to determine the distribution of leaf scald disease and this new pathotype/race of X. albilineans in Gabon and other geographical locations. References: (1) M. J. Davis et al. Plant Dis. 78:78, 1994. (2) P. Rott et al. Phytopathology 87:1202, 1997 (3) P. Rott et al. Mol. Plant-Microbe Interact. 24:594, 2011.

Polyphasic characterization of Xanthomonas axonopodis pv. allii associated with outbreaks of bacterial blight on three Allium species in the Mascarene archipelago.

Based on the number of new reports during the last two decades, bacterial blight of onion (Allium cepa) is considered an emerging disease. The causal agent, Xanthomonas axonopodis pv. allii, is pathogenic to several Allium species after inoculation, but outbreaks worldwide have been primarily reported on onion. We describe a unique epidemiological situation in Réunion Island, France, with concomitant outbreaks on three Allium species, onion, leek (A. porrum), and garlic (A. sativum). There was no host specialization within Allium spp. among strains associated with the three host species. Based on amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism, strains associated with these outbreaks in Réunion Island were highly related genetically to strains isolated from diseased plant samples and contaminated seed lots in the neighboring island of Mauritius, where the disease has occurred since 1984. All AFLP haplotypes were identified as X. axonopodis pv. allii based on polymerase chain reaction analysis using specific primers, biochemical tests, and/or pathogenicity tests. Two genetically related groups of strains (A and B) that can be distinguished by AFLP, differential utilization of three carbon sources, and xanthomonadin pigment production were detected initially after establishment of the pathogen. In less than 10 years after the establishment of the pathogen there was nearly an extinction of group A strains in Réunion Island, suggesting differences in fitness between strains in the two groups.

Quantitative and molecular epidemiology of bacterial blight of onion in seed production fields.

ABSTRACT Onion, a biennial plant species, is threatened by the emerging, seed-borne, and seed-transmitted Xanthomonas axonopodis pv. allii. Bacterial blight epidemics were monitored in seed production fields over two seasons. Temporal disease progress was different between the two seasons, with final incidence ranging from 0.04 to 0.06 in 2003 and from 0.44 to 0.61 in 2004. The number of hours with temperatures above 24 degrees C was the best descriptor for predicting the number of days after inoculation for bacterial blight development on inoculated plants. Fitting the beta-binomial distribution and binary power law analysis indicated aggregated patterns of disease incidence data. The beta-binomial distribution was superior to the binomial distribution for 97% of the examined data sets. Spatial dependency ranged from 5.9 to 15.2 m, as determined by semivariance analysis. Based on amplified fragment length polymorphism (AFLP) analysis, it was concluded that plots predominantly were infected by the inoculated haplotype. A single other haplotype was identified by AFLP in all plots over the 2 years, and its detection in the field always followed wind-driven rains. X. axonopodis pv. allii-contaminated seed were detected by semiselective isolation and a nested polymerase chain reaction assay at levels up to 0.05% when final disease incidence was 0.61. Contaminated seed originated from both diseased and asymptomatic plants.

New media for the semiselective isolation and enumeration of Xanthomonas campestris pv. mangiferaeindicae, the causal agent of mango bacterial black spot.

AIMS: Mango bacterial black spot, caused by Xanthomonas campestris pv. mangiferaeindicae, is a potentially severe disease in several tropical and subtropical areas. Data describing the life cycle of the pathogen are needed for improving integrated pest management strategies. Because of the important bacterial microflora associated with mango leaves, isolation of the pathogen is often difficult using nonselective agar media. METHODS AND RESULTS: A previously developed medium, BVGA, failed to inhibit several Gram-negative saprophytic bacteria, especially those belonging to Enterobacteriaceae. Two new semiselective media were developed. The selectivity of KC and NCTM3 media was achieved using cephalexin 40 mg l(-1), kasugamycin 20 mg l(-1) and neomycin 1 mg l(-1), cephalexin 100 mg l(-1), trimethoprime 5 mg l(-1), pivmecillinam 100 mg l(-1) respectively. Plating efficiencies ranged from 76 to 104% and from 78 to 132% for KC and NCTM3 respectively. CONCLUSIONS: The new media allowed the growth of X. campestris pv. mangiferaeindicae whatever its country of isolation. The pathogen was repeatedly isolated with these media from asymptomatic leaves sampled in growth chamber experiments. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides a description of new semiselective media, which should be valuable tools to study the ecology and epidemiology of X. campestris pv. mangiferaeindicae.

Polyphasic characterization of xanthomonads isolated from onion, garlic and Welsh onion (Allium spp.) and their relatedness to different Xanthomonas species.

Bacterial blight is an emerging disease that affects primarily onion, but also garlic and Welsh onion. The present study was undertaken to characterize the causative xanthomonad(s) by a polyphasic approach using a worldwide collection of 33 bacterial strains. Analysis of 16S rRNA gene sequence similarities indicated that the causal agent belongs to the campestris core in the genus Xanthomonas, which is in agreement with results of phenotypic characterization (analyses of carbon source utilization and fatty acid methyl esters). However, DNA-DNA hybridization, thermal stability of DNA reassociation and fluorescent amplified fragment length polymorphism analysis allowed the causal agent to be identified as a pathovar of Xanthomonas axonopodis.

Spatial and Temporal Analyses of Bacterial Blight of Onion Caused by Xanthomonas axonopodis pv. allii.

ABSTRACT Bacterial blight of onion is a severe disease, which emerged over the past decade in several onion-producing areas. This disease currently is observed in both the Old and New Worlds. Although the causative agent, Xanthomonas axonopodis pv. allii, is potentially seedborne, seed transmission and significance of seedborne initial inoculum for the development of bacterial blight of onion previously has not been assessed. This article describes experimental work designed to evaluate the biological importance of seedborne X. axonopodis pv. allii as an initial inoculum source, and examine the temporal and spatial dynamics of the disease. Over 3 years, outbreaks of bacterial blight of onion always were induced in experimental plots sown with naturally contaminated seed lots, with a contamination rate determined as 0.04%. Analyses of disease patterns indicated a likely seedborne origin for the inoculum associated with the early stages of epidemics. Spatial analyses performed with several statistical methods indicated aggregated patterns of disease incidence data. Primary foci enlarged over time, and a few distinct secondary foci sometimes were established after occurrence of wind-driven rains (with gusts up to 15 m s(-1)). Distances between primary and secondary foci ranged from less than 1 m (satellite foci) to 25 m. It remains possible that longdistance dispersal of inoculum was at least partly involved in the later stages of epidemics.

First Report of Anthurium Blight Caused by Xanthomonas axonopodis pv. dieffenbachiae in Reunion Island.

Xanthomonas axonopodis pv. dieffenbachiae is the causal agent of Anthurium blight, a severe systemic disease of Anthurium. Bacterial blight has been reported in most of the areas where Anthurium is cultivated, especially in Hawaii, California, Guadeloupe, Martinique, Jamaica, and Venezuela. This pathogen is also described on many genera of the Araceae family, e.g., Dieffenbachia, Syngonium, Philodendron, Caladium, Aglaonema, and Colocasia. In Reunion Island, Anthurium blight was first observed in 1997 during routine inspections in two nurseries on Anthurium andreanum plants imported from the Netherlands. Lesions consisted of water-soaked spots at the leaf margins surrounded by chlorotic or necrotic zones. Several necrotic lesions had coalesced to form large, dark patches that covered a large portion of the leaf. Some plants showed symptoms of systemic decay. The disease rapidly spread to other Anthurium plants in the nurseries, causing severe damage. Bacteria, which were consistently isolated from infected plants, were gram negative, yellow-pigmented, and mucoid. Carbon source utilization patterns (Biolog, Hayward, CA) were consistent with Xanthomonas sp. and the bacteria responded positively to monoclonal antibodies raised against X. axonopodis pv. dieffenbachiae (Agdia) in an enzyme-linked immunosorbent assay. Between 1997 and 1999, 114 isolates were collected from three main locations: the two primary infected nurseries and another one found contaminated later. Pathogenicity tests were performed on 8-month-old plants of A. andreanum cv. Carré by infiltrating the leaves with a suspension of bacteria (105 CFU/ml) using a syringe. Each strain was inoculated onto three young leaves (four inoculation points per leaf) on two plants. Control plants received sterile Tris solution. Plants were maintained in a growth chamber at 28°C (±1°C) with 95% (±5%) relative humidity and a photoperiod of 12 h. All 114 strains caused typical symptoms on Anthurium with the development of water-soaked spots near the inoculation point after 9 days, evolving into chlorotic and then necrotic areas after 20 to 24 days. No symptoms developed on control plants. Koch's postulates were completed by reisolating, from all the inoculated plants, bacteria that were again serologically identified as X. axonopodis pv. dieffenbachiae. Since 1997, control measures were adopted, consisting of the destruction of the infected plants and quarantining the contaminated nurseries, as well as surveillance intensification at the ports of entry to prevent new introductions.

Transpiration of a 64-year-old maritime pine stand in Portugal : 1. Seasonal course of water flux through maritime pine.

The transpiration, sap flow, stomatal conductance and water relations ofPinus pinaster were determined during spring and summer in a 64-year-old stand in Ribatejo (Portugal). The transpiration of the pine canopy was determined from sap flow or eddy covariance techniques. Canopy conductance values (g c) were estimated from inversion methods using eddy covariance or sap flow data, respectively, and from scaling-up methods using stomatal conductance values measured in the field and leaf area index (LAI) values. The transpiration was closely controlled by the stomatal conductance of pines (Ω was 0.05-0.15). For wet soil conditions, the various estimates ofg c showed reasonable agreement.g c peaked in the morning at 0.01 m×s-1, exhibited a midday depression and showed a secondary peak in late afternoon. This behaviour could be predicted simply on the basis of the stomatal sensitivity to air vapour pressure deficit. On a seasonal basis, monthly average values ofg c decreased from 4×10-3 m×s-1 in spring to 1.7×10-3 m·s-1 in late summer. Accordingly, the transpiration peaked at 3 mm×d-1 on wet soil in May. It decreased progressively during the summer drought to 0.8 mm×d-1 at the end of August. The minimal value of needle water potential was maintained at -1.9 MPa but predawn values decreased from -0.6 MPa in May to -0.9 MPa in July. It may have reached lower values in August. The amount of water stored in the trunk accounted for a 12% (10 kg×tree-1×day-1) of the daily transpiration in spring. The storage capacity of the canopy was within the same order of magnitude. The trunk storage increased to 25% (13 kg×tree-1×day-1) of the daily transpiration at the end of summer under drought conditions. The sap flow beneath the crown lagged accordingly behind transpiration with a time constant estimated between 26 min in spring and 40 min at the end of summer.