While there are documented host shifts in many bacterial plant pathogens, the genetic foundation of host shifts is largely unknown. Xylella fastidiosa is a bacterial pathogen found in over 600 host ...plant species. Two parallel host shifts occurred—in Brazil and Italy—in which X. fastidiosa adapted to infect olive trees, whereas related strains infected coffee. Using 10 novel whole‐genome sequences from an olive‐infecting population in Brazil, we investigated whether these olive‐infecting strains diverged from closely related coffee‐infecting strains. Several single‐nucleotide polymorphisms, many derived from recombination events, and gene gain and loss events separated olive‐infecting strains from coffee‐infecting strains in this clade. The olive‐specific variation suggests that this event was a host jump with genetic isolation between coffee‐ and olive‐infecting X. fastidiosa populations. Next, we investigated the hypothesis of genetic convergence in the host shift from coffee to olive in both populations (Brazil and Italy). Each clade had multiple mutations and gene gain and loss events unique to olive, yet no overlap between clades. Using a genome‐wide association study technique, we did not find any plausible candidates for convergence. Overall, this work suggests that the two populations adapted to infect olive trees through independent genetic solutions.
In Xylella fastidiosa, two parallel host shifts to infect olive trees occurred in Brazil and Italy, but the lack of genetic convergence in olive‐infecting strains across the two distinct clades suggests independent genetic solutions.
Summary
Citrus sudden death‐associated virus (CSDaV) is a member of the genus Marafivirus in the family Tymoviridae, and has been associated with citrus sudden death (CSD) disease in Brazil. ...Difficulties in the purification of CSDaV from infected citrus plants have prevented progress in the investigation of the role of this virus in CSD and an understanding of its molecular biology. In this work, we have constructed a full‐length cDNA clone of CSDaV driven by the 35S promoter (35SRbz‐CSDaV). Agrobacterium tumefaciens‐mediated inoculation of 35SRbz‐CSDaV in Nicotiana benthamiana plants enabled a fast recovery of large amounts of virions from the agroinfiltrated leaves, which allowed a better molecular characterization of CSDaV. In vivo analyses of mutant versions of 35SRbz‐CSDaV revealed the expression strategies used by CSDaV for production of the capsid proteins (CPs). We showed that CSDaV virions contain three forms of CP, each of which is generated from the same coding sequence, but by different mechanisms. The major CPp21 is a product of direct translation by leaky scanning from the second start codon in the subgenomic RNA (sgRNA), whereas the minor CPs, p25 and p23, are produced by direct translation from the first start codon in the sgRNA and by trans‐proteolytic cleavage processing derived from the p25 precursor, respectively. Together, these findings contribute to advance our understanding of CSDaV genome expression strategies. In addition, the construction and characterization of the CSDaV infectious clone represent important steps towards the investigation of the role of this virus in CSD and of its use as a tool for citrus biotechnology.
Homologous recombination affects the evolution of bacteria such as Xylella fastidiosa, a naturally competent plant pathogen that requires insect vectors for dispersal. This bacterial species is ...taxonomically divided into subspecies, with phylogenetic clusters within subspecies that are host specific. One subspecies, pauca, is primarily limited to South America, with the exception of recently reported strains in Europe and Costa Rica. Despite the economic importance of X. fastidiosa subsp. pauca in South America, little is known about its genetic diversity. Multilocus sequence typing (MLST) has previously identified six sequence types (ST) among plant samples collected in Brazil (both subsp. pauca and multiplex). Here, we report on a survey of X. fastidiosa genetic diversity (MLST based) performed in six regions in Brazil and two in Argentina, by sampling five different plant species. In addition to the six previously reported ST, seven new subsp. pauca and two new subsp. multiplex ST were identified. The presence of subsp. multiplex in South America is considered to be the consequence of a single introduction from its native range in North America more than 80 years ago. Different phylogenetic approaches clustered the South American ST into four groups, with strains infecting citrus (subsp. pauca); coffee and olive (subsp. pauca); coffee, hibiscus, and plum (subsp. pauca); and plum (subsp. multiplex). In areas where these different genetic clusters occurred sympatrically, we found evidence of homologous recombination in the form of bidirectional allelic exchange between subspp. pauca and multiplex. In fact, the only strain of subsp. pauca isolated from a plum host had an allele that originated from subsp. multiplex. These signatures of bidirectional homologous recombination between endemic and introduced ST indicate that gene flow occurs in short evolutionary time frames in X. fastidiosa, despite the ecological isolation (i.e., host plant species) of genotypes.
Citrus is a globally important, perennial fruit crop whose rhizosphere microbiome is thought to play an important role in promoting citrus growth and health. Here, we report a comprehensive analysis ...of the structural and functional composition of the citrus rhizosphere microbiome. We use both amplicon and deep shotgun metagenomic sequencing of bulk soil and rhizosphere samples collected across distinct biogeographical regions from six continents. Predominant taxa include Proteobacteria, Actinobacteria, Acidobacteria and Bacteroidetes. The core citrus rhizosphere microbiome comprises Pseudomonas, Agrobacterium, Cupriavidus, Bradyrhizobium, Rhizobium, Mesorhizobium, Burkholderia, Cellvibrio, Sphingomonas, Variovorax and Paraburkholderia, some of which are potential plant beneficial microbes. We also identify over-represented microbial functional traits mediating plant-microbe and microbe-microbe interactions, nutrition acquisition and plant growth promotion in citrus rhizosphere. The results provide valuable information to guide microbial isolation and culturing and, potentially, to harness the power of the microbiome to improve plant production and health.
Xylella fastidiosa, an economically important plant-pathogenic bacterium, infects both coffee and citrus trees in Brazil. Although X. fastidiosa in citrus is well studied, knowledge about the ...population structure of this bacterium infecting coffee remains unknown. Here, we studied the population structure of X. fastidiosa infecting coffee trees in São Paulo State, Brazil, in four regions where citrus is also widely cultivated. Genotyping of over 500 isolates from coffee plants using 14 genomic microsatellite markers indicated that populations were largely geographically isolated, as previously found with populations of X. fastidiosa infecting citrus. These results were supported by a clustering analysis, which indicated three major genetic groups among the four sampled regions. Overall, approximately 38% of isolates showed significant membership coefficients not related to their original geographical populations (i.e., migrants), characterizing a significant degree of genotype flow among populations. To determine whether admixture occurred between isolates infecting citrus and coffee plants, one site with citrus and coffee orchards adjacent to each other was selected; over 100 isolates were typed from each host plant. No signal of natural admixture between citrus- and coffee-infecting isolates was found; artificial cross-infection assays with representative isolates also yielded no successful cross infection. A comparison determined that X. fastidiosa populations from coffee have higher genetic diversity and allelic richness compared with citrus. The results showed that coffee and citrus X. fastidiosa populations are effectively isolated from each other and, although coffee populations are spatially structured, migration has an important role in shaping diversity.
The Asian citrus psyllid (ACP) Diaphorina citri, vector of 'Candidatus Liberibacter asiaticus' (CLas), the putative causal agent of citrus Huanglongbing (HLB), is controlled by application of ...insecticides, which, although effective, has resulted in serious biological imbalances. New management tools are needed, and the technique known as "trap crop" has been attracting attention. A potential plant for use as a trap crop in the management of the ACP is Murraya koenigii (curry leaf). However, for this plant to be used in the field, it needs to be attractive for the vector and must not harbor CLas. To verify the potential of curry leaf as trap crop for the management of HLB, we investigated the ability of D. citri to transmit CLas to M. koenigii, and to other test plants, including M. paniculata (orange jasmine) and cultivar Valencia sweet-orange seedlings. For the tests, the insects were reared on a symptomatic CLas-infected plant and allowed to feed on the three test plant species. The overall maximum transmission rate for the citrus seedlings was 83.3%, and for orange jasmine was 33.3%. Successful transmission of CLas by ACP to the curry-leaf seedlings was not observed, and it was treated as immune to CLas. Supported by the previous results that M. koenigii is attractive for ACP, these results indicate that curry leaf is an excellent candidate for use as a trap crop, to improve the management of the insect vector and consequently of HLB.
Xylella fastidiosa subsp. pauca is genetically diverse and has many vector species. However, there is limited information on vector specificity and efficiency for different sequence types (STs) ...within the pathogen subspecies. Both STs of X. fastidiosa and vectors differ in their associations with plants; therefore, assessment of vector competence should include the standardized vector acquisition ability of bacteria from artificial diets. This work aimed to adapt and validate an in vitro acquisition system for strains of X. fastidiosa that cause citrus variegated chlorosis, and to compare the transmission efficiency of STs of subsp. pauca by different species of sharpshooter vector. First, acquisition and transmission of ST13 by Bucephalogonia xanthophis and Macugonalia leucomelas was tested using an artificial diet with bacteria grown on minimum defined medium (X. fastidiosa medium) with or without 1% galacturonic acid (GA). Subsequently, four sharpshooter species (B. xanthophis, M. leucomelas, M. cavifrons, and Sibovia sagata) were compared as vectors of ST13 acquired from artificial diets, and four STs of subsp. pauca (11, 13, 65, and 70) were tested for acquisition and transmission by M. leucomelas. The artificial system allowed efficient acquisition and transmission of ST13 to plants, with no differences between the media tested. ST13 was transmitted more efficiently by B. xanthophis and M. leucomelas when compared with M. cavifrons and S. sagata. Different STs influenced acquisition and transmission rates by M. leucomelas. The differences in vector competence, despite the standardized acquisition system, suggest that ST-vector foregut or vector-plant interactions may influence bacterial acquisition, retention and inoculation by the insect.
The ecology of plant pathogens of perennial crops is affected by the long-lived nature of their immobile hosts. In addition, changes to the genetic structure of pathogen populations may affect ...disease epidemiology and management practices; examples include local adaptation of more fit genotypes or introduction of novel genotypes from geographically distant areas via human movement of infected plant material or insect vectors. We studied the genetic structure of Xylella fastidiosa populations causing disease in sweet orange plants in Brazil at multiple scales using fast-evolving molecular markers (simple-sequence DNA repeats). Results show that populations of X. fastidiosa were regionally isolated, and that isolation was maintained for populations analyzed a decade apart from each other. However, despite such geographic isolation, local populations present in year 2000 were largely replaced by novel genotypes in 2009 but not as a result of migration. At a smaller spatial scale (individual trees), results suggest that isolates within plants originated from a shared common ancestor. In summary, new insights on the ecology of this economically important plant pathogen were obtained by sampling populations at different spatial scales and two different time points.
Xylella fastidiosa is a plant pathogen bacterium that causes diseases in many different crops. In citrus, it causes Citrus Variegated Chlorosis (CVC). The mechanism of pathogenicity of this bacterium ...is associated with its capacity to colonize and form a biofilm in the xylem vessels of host plants, and there is not yet any method to directly reduce populations of this pathogen in the field. In this study, we investigated the inhibitory effect of N-Acetylcysteine (NAC), a cysteine analogue used mainly to treat human diseases, on X. fastidiosa in different experimental conditions. Concentrations of NAC over 1 mg/mL reduced bacterial adhesion to glass surfaces, biofilm formation and the amount of exopolysaccharides (EPS). The minimal inhibitory concentration of NAC was 6 mg/mL. NAC was supplied to X. fastidiosa-infected plants in hydroponics, fertigation, and adsorbed to organic fertilizer (NAC-Fertilizer). HPLC analysis indicated that plants absorbed NAC at concentrations of 0.48 and 2.4 mg/mL but not at 6 mg/mL. Sweet orange plants with CVC symptoms treated with NAC (0.48 and 2.4 mg/mL) in hydroponics showed clear symptom remission and reduction in bacterial population, as analyzed by quantitative PCR and bacterial isolation. Experiments using fertigation and NAC-Fertilizer were done to simulate a condition closer to that normally is used in the field. For both, significant symptom remission and a reduced bacterial growth rate were observed. Using NAC-Fertilizer the lag for resurgence of symptoms on leaves after interruption of the treatment increased to around eight months. This is the first report of the anti-bacterial effect of NAC against a phytopathogenic bacterium. The results obtained in this work together with the characteristics of this molecule indicate that the use of NAC in agriculture might be a new and sustainable strategy for controlling plant pathogenic bacteria.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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