Using the race 1 resistance phenotypes observed in our studies and California Strawberry Commission production statistics , we discovered that susceptible cultivars have been planted on 49-85% of the acreage in California over the last eleven years . That percentage has hovered between 55 and 59% since 2014. Hence, susceptible cultivars continue to be widely planted in California despite incontrovertible evidence that losses to the disease can be prevented by planting cultivars carrying one of the race-specific R-genes we identified . Over five years of screening plants that were either naturally infected or artificially inoculated with race 1 isolates of the pathogen, we have not observed visible symptoms on cultivars or other germplasm accessions carrying the dominant FW1 allele . Although private sector cultivars were unavailable for inclusion in our studies,seedling starter pot the prevalence of R-genes in publicly available germplasm collections and shared ancestry of public and private sector cultivars worldwide suggests that the same R-genes are widely found in private sector cultivars .
We anticipate that production in California will ultimately shift away from susceptible cultivars, particularly as the incidence of the disease increases and yield losses mount .The cloning and characterization of R-genes underlying race-specific resistance is important for developing an understanding of their function and interactions with the pathogen and building the foundation needed to engineer resistance through genome editing or other approaches . The I genes that confer race-specific resistance to F. oxysporum f. sp. lycopersici in tomato differ in durability and function and provide a model for future studies in strawberry . The I-2 gene had a signifcantly longer life span than the I gene, which was defeated in less than a decade subsequent to deployment . The durability differences of the tomato I genes have been attributed to differences in the dispensability of avirulence genes or mutations in avirulence genes that defeat known R-genes . The life spans of the race-specific R-genes we identified in strawberry are of course unknown; however, the sheer abundance and diversity of race 1 R-genes found in the wild relatives predict that sources of resistance to other races of the pathogen can be rapidly identified and deployed . The earliest reports of resistance to California isolates of the pathogen emerged when the disease initially surfaced in field experiments in California where well known cultivars were being grown .
We have since shown that the resistance phenotypes of those cultivars were mediated by FW1; hence, the FW1 gene has endured for at least 16 years to-date. Our search for diverse R-genes was partly motivated by the need to prepare for the havoc created by the inevitable evolution and emergence of novel pathogen races and inadvertent introduction of foreign races of the pathogen through infected plants or soil . Our findings suggest that the resistant germplasm accessions identified in the present study carry one or more dominant Fusarium wilt R-genes, and that race 1 R-genes are found in a wide range of heirloom and modern cultivars . The latter finding further suggests that race 1 resistance genes are found in domesticated populations worldwide, albeit often at low frequency because they have not been consciously selected, e.g., we previously showed that the frequency of FW1 was 0.16 in the pre-2015 California population and that FW1 originated in Shasta and other cultivars released in the 1930s , 70 to 80 years before Fusarium wilt was first reported in California . The phenotypic and pedigree databases we developed should expedite the identification and incorporation of Fusarium wilt resistance into modern cultivars . Our data suggest that a certain percentage of modern cultivars are bound to fortuitously carry Fusarium wilt R-genes. That was exactly what we discovered in the California population .
The race 1 R-genes we identified in cultivated strawberry are predicted to be a small sample of those found in the wild reservoir of genetic diversity . Our analyses of the pedigree records of heirloom and modern cultivars show that those R-genes were fortuitously introduced through early founders and survived breeding bottlenecks predating the late twentieth century emergence of this disease in strawberry . Their chance survival in individuals that dominate the ancestry of domesticated populations worldwide is noteworthy because artificial selection for resistance to Fusarium wilt was not consciously applied anywhere outside of Australia or Japan until 2015 when we initiated breeding for resistance to California-specific isolates of the pathogen . This suggests that genes conferring resistance to Fusarium wilt were fairly common in the founders, which is what our data showed—52% of the wild octoploid individuals screened in the present study were highly resistant to race 1 and predicted to carry dominant race 1 R-genes . Our analyses of pedigree records further suggest that many of the race 1 R-genes found in heirloom and modern cultivars have flowed through common ancestors and thus could be identical-by-descent . We cast a wide net in our original phenotypic screening experiments because the genetic basis of resistance was unknown before race-specific R-genes were discovered , knowledge was lacking to strategically narrow the search, and the frequency of resistance among accessions preserved in public germplasm collections was unknown. Our phenotypic screens were designed by assuming that we might be searching for a needle in a haystack, primarily because phenotypic screens in tomato and other plant species had shown that genes conferring resistance to Fusarium wilt were uncommon or only found in wild relatives, e.g., the Fusarium wilt R-genes in cultivated tomato were transferred from wild relatives . Although the domestication and breeding histories of tomato and strawberry are quite different, the frequencies of Fusarium wilt resistance among accessions of wild relatives are similar. Fifty-two to 57% of the individuals we sampled from wild populations of F. chiloensis and F. virginiana were resistant to races 1 and 2, which are comparable to the percentages reported for race-specific R-genes in the wild relatives of tomato .Wild relatives could certainly become an important source of R-genes in strawberry breeding going forward; however, the prevalence of R-genes in modern cultivars circumvents the need to introduce alleles from wild relatives or exotic sources, which has often been necessary for the development of Fusarium wilt resistant cultivars in tomato, cotton, and other plants .
The wild relatives of many of the agriculturally important species impacted by this pathogen carry chromosome rearrangements or structural DNA variation that impedes gene flow and the recovery of recombinants, e.g., the tomato I genes have been introgressed from wild relatives with interspecific structural variation that suppresses recombination and causes the persistence of unfavorable alleles through linkage drag . Notably in strawberry, the octoploid progenitors are inter-fertile and have highly syntenic genomes with no known or apparent barriers to gene flow or suppressed recombination in wide crosses . This does not eliminate the linkage drag problem altogether but simplifes the challenge of purging unfavorable alleles introduced by exotic donors in wide crosses . Moreover,round nursery pots cultivated strawberry has emerged from only 250 years of domestication in interspecific hybrid populations between wild relatives and thus has not experienced population bottlenecks on a scale similar to wheat, tomato, and other staples that have undergone 7000 to 10,000 years of domestication.Both high and low acidity titrant solutions HI84532-51 and HI84532-50 were used to titrate high acid and low acid pomegranate juices, respectively. Juice samples were autotitrated with OH- -based titrant solutions to an endpoint pH of 8.1 to calculate the percentage citric acid in solution. One sample was run per fruit juice sample. Solvents, chemicals, and reagents used to measure total phenolics and antioxidant activity of the pomegranate juice were of analytical grade. Solvents used were ethanol and methanol . Chemicals used were gallic acid and 2,2-diphenyl-1-picrylhydrazyl . Reagents used were Folin-Ciocalteu reagent , and sodium carbonate .Juice TP were measured utilizing the Folin-Ciocalteu method as described by Tezcan et al. . For TP, 3 µL of 100% juice supernatant was added to 297 µL 6:4 methanol:deionized water into a 4 mL cuvette. Then 1.5 mL of 10-fold-diluted Folin–Ciocalteu reagent was added and left to react for 3 min. After this reaction period, 1.2 mL of 7.5% sodium carbonate was added to the cuvette and the sample was left in the dark for 90 min. The cuvette was transferred to a Biochrom Ultrospec II spectrophotometer and the absorbance was measured at 765 nm. A standard curve was plotted and the linear equation used to quantify TP in gallic acid equivalents. All results for TP were expressed in mg/L of gallic acid equivalents . Juice TP samples were measured in triplicate and averaged due to the variation within samples. The 10-folddiluted Folin–Ciocalteu reagent was made by measuring 25 mL of 100% Folin–Ciocalteu and pouring it into a 250 mL volumetric flask and bringing up the volume to 250 mL using DI water. The 7.5% sodium carbonate solution was formulated by dissolving 7.5 g of powder sodium carbonate in 100 mL of DI water and mixing with a magnetic stir bar under low heat. All parameters were analyzed with Analysis of Variance . When ANOVA indicated significant differences, post-hoc comparisons were performed utilizing Tukey’s honestly significant difference with an experiment wise error rate of α = 0.05. Relationships between all parameters were analyzed using linear regression , with correlations among parameters determined using general regression with Minitab Software, version 16 .
There were differences among the ten pomegranate cultivars for fruit weight . Phoenicia and Wonderful fruit were among the heaviest, and the fruit weights of these cultivars were significantly different from those of Ambrosia, Eversweet, and Haku Botan. The lightest fruit by weight was Eversweet, and several cultivars had significantly lighter fruit than Wonderful. Fruit weight typically increased over time during both seasons for both a given cultivar and regardless of cultivar. During 2015, fruit weight slightly decreased when pooling weights of all cultivars for that year because some of the heaviest cultivars were not available for that harvest date. To mitigate issues with an unbalanced design, we did not include November fruit in internal fruit quality analysis. Fruit weight was positively correlated with fruit diameter, but not correlated with fruit length. Antioxidant activity was weakly and negatively correlated with fruit weight . There were no other notable or strong correlations for fruit weight. There were differences among the ten pomegranate cultivars for fruit diameter and fruit length . When pooling data, no cultivars had fruit that were significantly larger than Wonderful. For fruit length, fruit of Phoenicia and Wonderful were longest and Eversweet had the shortest fruit of all cultivars. Fruit diameter increased from September to October, but then decreased in November due to lack of availability of large fresh market fruit this late in the season. Most fruit were destroyed by weather, fruit splitting, and pest damage by mid-November for many cultivars, so mean diameter decreased from October to November. Fruit length also increased from September to October, but then decreased in November due to lack of fresh market fruit available from those trees by the end of the season. There were no differences between years for fruit weight, diameter and length when data were pooled to evaluate effect of year on these traits. ‘Ambrosia,’ and ‘Green Globe’ had the heaviest arils, which were significantly different than ‘Wonderful’ . Haku Botan, an ornamental cultivar, had the least heavy arils, which were also significantly lighter than Wonderful. For weight of all arils, ‘Golden Globe,’ ‘Parfianka,’ ‘Phoenicia,’ and ‘Wonderful’ had the greatest weight and ‘Eversweet’ and ‘Haku Botan’ had the least total aril weight on average. ‘Green Globe’ had the highest fruit edible fraction and this was significantly greater than ‘Wonderful,’ having over 15% more edible fraction in the fruit than ‘Wonderful.’ ‘Ambrosia,’ ‘Golden Globe,’ ‘Parfianka,’ and ‘Phoenicia’ also had significantly higher fruit edible fraction than ‘Wonderful.’ ‘Haku Botan’ and ‘Loffani’ had the least fruit edible fraction, with less than 50% of the fruit weight being edible. The only other cultivar with less than 50% fruit edible fraction was Wonderful. There were no notable correlations for weight of 100 arils, weight of all arils, and fruit edible fraction with any other variable. Weight of 100 arils increased from September to October, but then leveled out when comparing October to November . There were significant differences among cultivars for °Brix .