The structure and function of the global citrus rhizosphere microbiome

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

Parallel host shifts in a bacterial plant pathogen suggest independent genetic solutions.

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

Spatial Genetic Structure of Coffee-Associated Xylella fastidiosa Populations Indicates that Cross Infection Does Not Occur with Sympatric Citrus Orchards.

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

Spatial Genetic Structure of Coffee-Associated Xylella fastidiosa Populations Indicates that Cross Infection Does Not Occur with Sympatric Citrus Orchards.

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

Rescue of Citrus sudden death-associated virus in Nicotiana benthamiana plants from cloned cDNA: insights into mechanisms of expression of the three capsid proteins.

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

Rescue of Citrus sudden death-associated virus in Nicotiana benthamiana plants from cloned cDNA: insights into mechanisms of expression of the three capsid proteins.

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

Citrus in the world

This report provides an assessment of citrus health globally. Citrus trees are a perennial crop grown for its fruits, which are one of the key sources of vitamin C in human diets (Liu et al 2012), a source of simple sugars, dietary fibre and secondary metabolites (Lv et al 2015), and a key ingredient in juices, jams, jellies and marmalades. Citrus fruits and by-products from juice production are also used to extract essential oils and citric acids used in the food, cosmetics and pharmaceutical industries (Kaur et al 2021; Panwar et al 2021). It is estimated that around 2/3 of citrus fruits produced worldwide are consumed as fresh fruit, and the remaining 1/3 is used mainly for juice production and some industrial purposes (Liu et al 2012)

Rescue of Citrus sudden death‐associated virus

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 and Xylella fastidiosa Host-Pathogen Associations in South America.

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