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 .A total of 6,236 SNPs were detected as being significantly associated with the host Vitis using the Bonferroni correction for multiple hypothesis testing with p-values <0.05. However, this still does not exclude the possibility of significance via phylogenetic proximity across the tree. Given the few clades of Vitis-associated bacteria, this dataset did not offer the power to use the most conservative method used by Scoary, the worst pairwise comparison p-value, which would identify only SNPs that have arisen independently across the phylogeny. However, data presented here , show genes that have a significant corrected P-value, and also have a best pairwise comparison P <=.125, showing some indication of independent emergence. 22 SNPs fit those criteria. Out of those 22, there were 9 genes in which several SNPs within them emerged as significant, likely due to linkage disequilibrium. Amongst the then total of 13 genes identified with significant SNPs, 5 are only identified to the level of hypothetical protein. Most genes that had multiple SNPs detected as significant also had identical frequencies of those SNPs across the populations, showing high linkage of those sites. Due to this, only one SNP is shown per gene and included in the enrichment tests. The 8 non-hypothetical significant genes are azu , carA, , clpP , nadD , mutY , ubiJ , exbD_1/2 , IpxD3/4 . KEGG orthologies show that 5 of the 8 identified and named genes are classified in the metabolism family in terms of their molecular functions . All identified SNPs were present at lower frequencies in strains from Vitis than other hosts, square black flower bucket wholesale with a mean frequency of 0.047 in Vitis derived strains and a mean frequency of 0.49 in non-Vitis derived strains.

Gene gain and loss was also tested using Scoary for host associations and the same pvalue corrections to go from 473 genes with Bonferroni p <0.05 to 37 genes that also had a best pairwise comparison p <= 0.125 . Out of the 37 identified genes, 33 are only identified as hypothetical proteins. The four named genes are xerC_1/2 , hcaB , mdtA_1/2/3 , and a cluster identified using Panaroo that includes the three genes CnrA , swrC_2 , and acrF_2 .These genes each belong to a different KEGG orthology .Information about X. fastidiosa ssp. fastidiosa infections in coffee in Central America is still limited – little symptomatology is associated with the disease; however, there are persistent infections in the field . The results of this work suggest that the host jump included adaptation of the X. fastidiosa strains to Vitis and lessened the ability of the U.S. strains to infect C. arabica. However, the ability for the pathogen to move throughout the C. arabica plants’ xylem vessels demonstrates a higher infectivity than expected in a fully resistant plant. Movement of the strains away from the inoculation point was observed, which shows the ability of the bacteria to successfully degrade the pit membrane, which is often a barrier to colonization. However, in contrast to that finding, there was a reduction of positive-testing C. arabica plants over the course of the year after the inoculation. This demonstrates a lower infectivity in C. arabica than the strains have to V. vinifera where infections in a greenhouse are sustained post-inoculation. This could demonstrate a reduction in the ability to create chronic infections in C. arabica by the Vitis-adapted strains. It is possible that the chronic infections of X. fastidiosa in C. arabica plants observed in situ may be caused by high rates of re-inoculation by insect vectors rather than strain level variation in infectivity. That is not the case for ssp. fastidiosa in V. vinifera, just one inoculation is sufficient for high virulence, and only cold winter temperatures have been known to cure infections that are otherwise chronic . The virulence of the U.S. strains to C. arabica is not as high as to V. vinifera, as shown by the lack of severe symptoms in C. arabica.

Given that, this study does not offer evidence that the California ssp. fastidiosa strains are generally more virulent than the ancestral strains in Central America. While not a likely scenario, there is a possibility that instead of experiencing adaptation to a specific host, the introduced strains became generally more virulent, which has been hypothesized about the globally spreading ssp. pauca strain infecting olive in Italy . In this paper, we present hypervirulence as a possible scenario, but as it is not supported by the data, we were able to rule it out. Symptom development in infected C. arabica plants consists of minor stunted growth, compared to the severe leaf scorch, matchstick petioles, shriveled fruit, and often plant death that occurs in V. vinifera. There are records of virulence of X. fastidiosa in C. arabica, such as evaluation of ssp. pauca in Brazil. In inoculation experiments with those strains, C. arabica may still develop symptoms slowly, and the proportion of positive plants does also reduce, however after 8 months the percentage positive was around 30% , not entirely dissimilar to the results from this project, which extends some uncertainty about this system. All methods used detected genetic signatures of adaptation, and many genetic candidates were identified by multiple methods. We were able to identify genes and SNPs associated with the host Vitis as well as genes under positive selection in strains isolated from Vitis. These genes included many hypothetical proteins, however genes with known functions pertaining to infections by X. fastidiosa were also identified.The genes identified by multiple methods included those whose functions in X. fastidiosa have been previously investigated, while many still have unknown functions . ClpX, the gene for the ATP-dependent protease ATP binding subunit, was previously identified as being upregulated 4x during induction of biofilm condition , a physiological state that is vital for virulence and vector colonization . Mutations in the copper related gene copA have been found to drastically alter copper tolerance in X. fastidiosa, which is vital for agricultural survival given the frequency of copper in treating fungal infections in the vineyard, a fungicide that has been in use since the 18th century . It is conceivable that after a host jump into a vineyard, it would be necessary for pathogens to survive higher levels of copper exposure. DegP has been found to be upregulated upon heat shock in X. fastidiosa . TolB encodes for a translocation protein involved in membrane integrity and has been shown to be important for biofilm development, an important aspect of pathogenicity . These are among a suite of other genes that are both hypothetical proteins, or just understudied in X. fastidiosa but show evidence of being involved in the process of this climate and host shift.

Now that these genes have been identified, they are prime candidates for targeting in future experiments to determine their effects on host range and climate adaptation. This study also includes one hypothesis that lies outside the general narrative of the introduction event, namely the evaluation of infectivity of ssp. multiplex towards C. arabica, which has not been tested before. While in California, ssp. multiplex has never been found infecting grapevine in the field, it has been shown to generate non persistent infections, similar to what we observed in C. arabica, in the greenhouse . Recently, infections of grapevine by ssp. fastidiosa have been detected in the field in Virginia . While the ssp. multiplex infections were not highly virulent, plastic square flower bucket they were just as persistent as the ssp. fastidiosa strains. All three main subspecies of X. fastidiosa are able to infect C. arabica to some degree . In conclusion, we have identified a suite of genes that are related to a host switch to Vitis with a corresponding reduction in the ability to infect an ancestral host. These data support the hypothesis that the shift was not a host range expansion of the subspecies, but a reduction of ability to infect a former host while optimizing the ability to infect a new host species.The results of in-situ experiments can differ greatly from those conducted in controlled laboratory conditions, especially in applied biological systems. In pathology specifically, there are often ethical and regulatory hurdles that prevent the introduction of disease-causing pathogens into naïve systems, whether it is for human health, food security, or in natural systems. Researchers are therefore typically constrained to using controlled or model systems that are ethically, financially, and logistically feasible. For example, mice are often used to understand human disease, and quarantine greenhouses are used to study plant disease . These proxy systems are imperfect, as we intuitively know that there are differences between a mouse and a human, so there are differences between the potted plant on your windowsill and the towering redwoods of California. These differences are intuitive, but also biological as the effects of interactions between organisms and their complex environments are impossible to recreate, and these environmental and physiological differences directly affect the outcomes of research in plant pathology . The chances to experimentally infect plants in accurate conditions are few and far between, so rare opportunities provide critical research windows. One such system that suffers from a lack of realistic experimental control is Pierce’s disease of grapevines , a persistent burden to the vineyards of California since the introduction of the etiological agent into North America in the late 1800s . The pathogenic bacterial strains that colonized Vitis vinifera and cause PD have since spread to Taiwan, Europe, and the Eastern United States . Although the relationship between Xylella fastidiosa and PD has been understood since 1978 , this study is the first to document the progression of symptoms in mature commercial grapevines under field conditions over the course of several years with a known inoculation time and location on the plant. Despite the challenges of conducting this work in-situ, predominantly concern from the proprietors about infection spreading, we have made insights that counter much of the classic understanding of this disease system and deepen our understanding of symptom development and bacterial multiplication and movement in PD. Several studies have documented PD, which collectively create expectations for the progression of this disease. The pathogen is known to move through mature grapevines at least fast enough to enter the cordon from the shoot within the first year of infection . However, given that removal of the cordon does not reduce disease severity in subsequent years , the pathogen must be moving further into the plant, presumably into the trunk during the first year. It is also well established that there are differences in response to X. fastidiosa infection based on Vitis vinifera cultivar, with some cultivars more susceptible than others . Symptoms are presumed to first appear late in the season that infection occurred, except in especially susceptible cultivars, such as Chardonnay, in which they can arise sooner. Characteristic symptoms have been described as leaf scorch, uneven lignification of the shoots, shriveled berries, and “matchstick” petioles, a situation in which the leaf blade becomes detached from the petiole. In this system, disease symptoms and pathogen infection do not always persist through the winter, a phenomenon called overwinter curing. However, the mechanism for recovery has not yet been determined, and current explanations range from pathogen temperature susceptibility to plant defensive responses. Expectations for overwinter curing are both temperature and cultivardependent, and in Napa Valley, the expected recovery rate for early-season inoculations is around 30% . However, such data may not translate to plants grown in the vineyard for various biotic and abiotic reasons. Greenhouse conditions do not account for the complex nutritional, pest, and other biotic  and abiotic pressures of the field , and greenhouse assays of grapevines are typically conducted with an excised shoot, rather than a mature plant. Observational studies are similarly limited because they often miss aspects such as recovery, asymptomatic infections, or unexpected symptoms by not relying on controls, but instead seeking out expected symptoms. These studies have given us a view on characteristics of PD and its progression that we were able to experimentally test.