The marginal ancestral state likelihood estimates of each host for all internal nodes of the ML phylogenetic trees were calculated using the re-rooting method of Yang et al. in the R package phytools , and mapped using the package APE . This method uses the phylogeny of extant taxa to reconstruct ancestral traits of extinct ancestors by analyzing phylogenetic parameters , along with a model of nucleotide substitution, to build posterior probabilities of character states at each interior node by randomly re-rooting the tree at each internal node and calculating the probability of observing the extant distribution of traits over all possibilities of that internal node character identity. The ML estimates at each internal node were calculated based on both the equal rates transition model and the symmetrical rates transition model . The fit of the two models to the data was compared using the Akaike information criterion and can be seen in supplemental table . Scoary was used to test if the pan-genome was correlated with hosts at either the super-order scales or the genus scale by conducting a Fisher’s Exact Test . FET measures the association of each gene in the pangenome to a trait of interest, plastic planters which in this case is plant host. While FET requires no association between datapoints, Scoary uses a phylogeny in order to remove lineage specific interdependencies and corrects the p-value based on those interdependencies.
Significance was evaluated by the “worst pairwise comparison P”, for the phylogenetic corrections, not the naïvep-values from FET. Individual analyses were conducted to test for correlation of gene presence and absence with each of the 29 coded host groups .California is the center of American fruit, vegetable and nut production and is a globally important exporter of plants . As a hub of international plant trade, California has been both a source and a sink for the introduction of novel phytopathogens across the globe. Xylella fastidiosa ssp. fastidiosa is a bacterial pathogen that impacts grapevines and almonds in California. X. fastidiosa subspecies fastidiosa, was likely of Central American origin and possibly introduced via coffee imports to the United States in the 1800s . After its introduction into the U.S., the pathogen has continued to spread, including recent introductions into Spain and Taiwan . In California, outbreaks of X. fastidiosa ssp. fastidiosa have been devastating to the grape industry. In contrast, infections have not caused notable issues in the agriculturally important regions of Central America, where the ssp. has likely existed in the ecosystem for at least thousands of years . This is not a universal trait of X. fastidiosa in C. arabica as opposed to Vitis. In Brazil, X. fastidiosa ssp. pauca infections reduce C. arabica yields, despite the pathogen’s long history in the region . X. fastidiosa is complex in its strain specificity to various host plants, with many conflicting examples of host susceptibility in geographic areas. Nonetheless, documenting host susceptibility to different subspecies and strains of the pathogen is vital. Clarifying the process of host jumps in X. fastidiosa is urgent for global agricultural security, as new outbreaks continue to put plant health at risk. While it is likely that X. fastidiosa ssp. fastidiosa is broadly present in Central America, currently only Costa Rica has reported this pathogen.
Here we refer to “Costa Rican” strains as our representative Central American strains, although there is certainly much larger diversity of X. fastidiosa in the region. X. fastidiosa ssp. fastidiosa frequently infects C. arabica in Costa Rica without substantially impacting production, although subtle leaf curling symptoms correlate with X. fastidiosa infections . In Costa Rica, X. fastidiosa ssp. fastidiosa also infects periwinkle , guava , and avocado . While symptoms have been reported in these hosts, disease severity and progression have not been thoroughly evaluated. Some strains of X. fastidiosa ssp. fastidiosa have been detected in Vitis in Costa Rica, where V. vinifera is not commonly grown but only sequence-type data was collected for these strains. However, a recent study found that related strains from Costa Rica did not cause infections in Vitis . Given these results, infectivity and virulence by the same strain may differ between Vitis and C. arabica. A clear phylogenetic delineation of pathogenicity, which requires extensive crossinoculation experiments, has not yet been determined. This study begins to test the adaptation of the U.S.-introduced clade to Vitis cross-inoculation and computational methods. Within the genus Vitis, there is wide genetic diversity conferring variation in both tolerance and resistance to Pierce’s disease , the disease caused by X. fastidiosa ssp. fastidiosa infections in grapevines. Differences are seen between species such as the naturally tolerant V. arizonica and Muscadinia rotundifolia, and the susceptible V. vinifera . This variation has recently been used to hypothesize that there might have been PD present in the U.S. for longer than has been estimated by all evolutionary data . But disease susceptibility also varies largely within V. vinifera , an intensely bred species that includes a wide and diverse range of cultivars. In contrast to crespera in Costa Rica, PD in the United States is quite virulent, persistent, and economically damaging. Infection with the pathogen causes severe symptoms such as stunting, leaf scorch, shriveled fruit, and eventual plant death in many cases . There are several additional adversely impacted hosts of species within the introduced clade. Of economic importance is almond suffering Almond Leaf Scorch . Other hosts of strains in the introduced clade include maple , plum , ragweed , sweet cherry , and western redbud . C. arabica is a unique host for X. fastidiosa. It has widespread documented infectivity coupled with non-deadly symptoms, and it was likely the source of two economically devastating introduction events: to the United States in grape and to Italy in olive . Nonetheless, C. arabica is generally understudied as a host plant. Data from Brazil show that C. arabica and Citrus strains of X. fastidiosa ssp. pauca do not cross-infect , showing variation in host specificity. However, C. arabica is also a host of at least two of the three major subspecies of X. fastidiosa, showing broad susceptibility. It has been hypothesized that strains of X. fastidiosa do not need to undergo many genetic changes in order to infect C. arabica, potentially making it a host susceptible to low-cost host jumps . While ssp. pauca and ssp. fastidiosa have both been documented to infect C. arabica, slow progression and low virulence have led C. arabica to be described as a “latent carrier” . So far, research has shown that the strains able to infect C. arabica are quite diverse . The infectivity of X. fastidiosa ssp. multiplex has never been tested in C. arabica. Subspecies multiplex is native to the south-eastern United States and is present throughout the U.S., South America, and Europe . However, the advent of coffee production in California coupled with historic documentation of ssp. multiplex in Argentina, Brazil, and Paraguay, call for investigation of whether C. arabica can serve as a host to ssp. multiplex . Upon introduction to the U.S., X. fastidiosa was exposed to new hosts, climate, vectors, and agricultural practices alongside a rapidly growing agricultural industry. While it is likely that the introduced strains were exposed to selective pressures that increased specificity to the conditions in the U.S., mainly climate and host, plastic plant nursery pot that is not the only possibility. It is also plausible that instead of adapting to the specific pressures in the new environment, the introduced population of strains became generally hypervirulent.
This could explain the high level of virulence towards V. vinifera in the U.S. compared to the lower virulence observed in Costa Rica. We investigated both neutral and adaptive changes during the process of naturalization for this pathogen using whole genome sequences.In March 2022, 120 coffee plants were divided into equal groups and inoculated with either a buffer control or one of four CA strains of X. fastidiosa, ssp. fastidiosa: Napa1 , ALS17T5 , Je115 or subspecies multiplex ALS15T2 , or the sterile succinate-citrate-phosphate buffer control . While Napa1 and Je115 are both isolated from V. vinifera, they are from different climates and clades of X. fastidiosa, and might have experienced distinctive selective regimes . ALS17T5 was isolated from a symptomatic almond plant and is in one of several clades of strains infecting only almond trees, however this clade is nested within other clades that infect Vitis . Disease-free C. arabica plants were donated from Frinj coffee company for this experiment. As positive controls, 20 V. vinifera cv. Chardonnay were inoculated as well as 50 Helianthus annus. V. vinifera is known to be highly susceptible to subspecies fastidiosa but not subspecies multiplex, while Helianthus annus is susceptible to both subspecies . Cell suspensions were prepared by suspending week-old cells grown on solid medium in SCP buffer just prior to inoculation. Each suspension was made by scraping 10 streaks of 20µL into 1 mL of buffer. Inoculations were conducted using 2 10µL beads of inoculum and a 00-size entomological pin used to pin prick through the bead of cell suspension several times until the inoculum absorbed into the plant xylem. Inoculations were conducted on small plants with typically 3 full-leaf pairs, and two inoculum beads were placed just above and below the center leaf pair. Plants were not waThered the morning of inoculation to optimize absorption of the inoculum into the xylem vessels. Symptom measurements took place in August, September, October, December, 2022, and January and April 2023, following the March 2022 inoculation. In August and September 2022, all internode lengths along the main stem of the plant were measured along with the heights of each plant. In October, December, January, and April, only the total plant heights were measured to detect stunting. Plants were also visually assessed for foliar scorching.Control and experimental plants were tested for the presence of X. fastidiosa via qPCR or culturing. DNA was extracted using a DNeasy plant mini kit and then quantified using qPCR with a primer pair targeting the gene encoding RecF, RecF1_F+R; for qPCR protocol see Sicard et al. . qPCR was run in duplicate with positive and negative controls on each plate; all samples with Ct values of 37 or higher were considered “undetected” and were considered negative. Culturing was conducted using approximately 0.1 grams of petiole tissue, or the entire petiole and midrib of a C. arabica leaf. Samples were surface sterilized, chopped, ground in a Polytron, and plated on PWG media using the method from Hill and Purcell . On April 3 rd , 2022, petioles that were directly above the inoculations site were sampled from all 50 sunflower plants and X. fastidiosa populations were measured via both qPCR and cell culturing. On June 16th , 2022, V. vinifera plants were sampled from and also visually assessed for symptoms. On July 14th , one leaf from the 2 nd leaf pair above the inoculation point was used for detection via qPCR. On August 1 st, ,15th , and 18th , one leaf from the 2 nd leaf pair above the inoculation point was cultured from each C. arabica plant. On October 4 th , 2022, the opposite leaf was taken for qPCR. While Vitis is not as susceptible infections from ssp. multiplex, typically there is some detectable infection, however those infections have 10-100 fold lower population sizes than infections by ssp. fastidiosa . In February – April 2023, we cultured from the leaves using a different method due to loss of leaves from the ~ 8 cm above the inoculation point. The lowest 8 leaves were collected, and the petioles were pooled for sterilization, tissue grinding, and plating.All analyses were performed using R statistical software version 4.2.3 . Linear mixed models were built using the package lme4 to add a random effect to account for repeated sampling of the plants over the course of the experiment . As analyses of virulence, we tested the effects of variety and the interaction between treatment and sampling date on both internode length, and separately on plant height , each with a random effect of plant ID using the lmer function of lme4. As an analysis of infection persistence via detection assays, we tested the effects of variety and the interaction between treatment and sampling date on the count of plants that tested positive using a generalized linear mixed model with binomial error using the package glmmTMB and the function glmmTMB .