Eight of the nine markers showed highly significant association with die back resistance, consistent with the Tvr1 gene being located in this region. Although the threshold for declaring association significant was set at p < 0.001, most of the associations were significant at p ≤ 0.00001. The only exception was marker Cntg4252, where the most significant association reached only p = 0.0042. The low association between SNPs at this marker and die back resistance was somewhat unexpected, since Cntg4252 co-segregated with the resistance allele in the × Salinas mapping population. While unexpected, it is not uncommon that markers closely linked with a trait in a mapping population do not show association when tested on a set of diverse accessions. This problem is well documented in potato, where markers linked to the Gro1 and H1 resistance genes in the mapping population were tested on 136 unrelated cultivars. The Gro1-specific marker was not correlated with the resistance phenotype, while H1-specific marker was indicative of resistance in only four cultivars. A similar example can be shown for lettuce, where markers most tightly linked to the cor resistance gene were the least useful for diagnostic when tested in a large collection of cultivars. There are several other examples of markers tightly linked to resistance genes, but whose use present problems in material different from the original in which they were identified.Therefore, an important requirement for any molecular marker used in MAS is not just its applicability in a specific cross, but its association in a wide gene pool. From SNPs that were significantly associated with die back resistance, the best fit was observed for those located in marker Cntg10192. This is the second of two markers, the other being Cntg4252, that co-segregated with the resistance allele in the mapping population. It is intriguing that one of the two markers co-segregating with the Tvr1 allele in the mapping population showed no significant association in a set of diverse accessions,30 litre plant pots bulk while the other showed a perfect match. Although these two markers were not separated in the intraspecific population, the linkage map developed from the Salinas × UC96US23 cross indicates that they are 1 cM apart.
Therefore it is possible that testing more RILs from the intraspecific population would separate the two markers and Tvr1. Association of SNPs from marker Cntg10192 with the resistance allele was validated in a larger set of 132 diverse accessions from several horticultural types. The marker-trait association was observed not only in L. sativa, but also in two L. serriola accessions included in the study. However, while the susceptible haplotype is identical in both species , the resistant haplotypes are different . To investigate the relationship between Tvr1 and the resistance observed in L. serriola, we screened 119 F8 RILs from the Salinas × UC96US23 population for resistance to die back. If Tvr1 and the resistance locus from UC96US23 were distinct and unlinked, approximately 25% susceptible offspring would be observed. However, since all RILs were resistant to the disease , we concluded that the resistance locus in UC96US23 is either allelic or linked to Tvr1. The same conclusion was reached for the resistance locus in the primitive romaine-type accession PI491224. The three resistance loci are associated with three distinct haplotypes; resistance in cv. Salinas with R1, in PI491224 with R2, and in UC96US23 with R3. Even though all 200 L. sativa accessions from the two testing sets showed the same haplotype-resistance association, it is unlikely that the EST from which this marker was derived is directly involved in die back resistance. A search for protein similarity in the NCBI database indicates that Cntg10192 is similar to the copper ion binding protein from castorbean and the plastocyanin-like domain-containing protein from Arabidopsis . The annotated functions of these two proteins do not imply an obvious role in plant-pathogen interactions.Moreover, the two substitutions at marker Cntg10192 that are the most significantly associated with die back resistance are synonymous, coding the same amino acid. Assuming that marker Cntg10192 is not directly involved in the resistance, it is probable that a recombinant genotype will eventually be identified. On the other hand, marker-trait associations can be very strong between some tightly linked alleles. For example, Rick and Forbes [51] documented linkage between allozyme Aps1 and tomato resistance gene Mi that did not break in as many as 30 backcross generations. Chromosomal linkage group 2 contains a large cluster of resistance genes that confer resistance to downy mildew and lettuce root aphid.
However, the Cntg10192 marker is well separated from thiscluster on the Salinas × UC96US12 map. Moreover, Tvr1 is one of the few resistance genes that was not at a genetic position coincident with any type of candidate resistance gene so far mapped in lettuce. Thus, it is possible that Tvr1 is different from the common types of pathogen recognition genes.We used HRM to directly detect sequence variations in PCR amplicons. High-resolution melting curves were recorded by the slow and steady heating of PCR products in a Light Scanner instrument.The method worked well for most of the analyzed markers, however, in a few cases, alleles could not be distinguished. When this occurred, we applied two alternative approaches to increase sensitivity through heteroduplex formation. In one approach, the heteroduplex formation was facilitated through mixing of samples prior to PCR. For example, if one sample contained DNA from cv. Salinas only, the other one would contain a mix of DNA from both cv. Salinas and Valmaine. The second alternative used an unlabeled probe 20 bp to 35 bp long that was designed for the region carrying the SNP. The probe was included in the PCR mix prior to cycling but was not consumed during amplification due to 3′ block. Genotyping was accomplished by monitoring the melting of probe-target duplexes post-PCR as described in Light Scanner manual. Both of the above alternatives improved allele detection; however, the probe-target duplex approach appeared to be more sensitive.Emerging plant diseases represent a continuous threat to economically important crop plants. Pandemics that affect supplies of grains and other annual subsistence crops are of particular concern . Diseases of perennial fruit trees are often very important for producers, due to the high costs of establishing intensively cultivated orchards and the extended amount of time needed to recover from outbreaks of diseases for which no effective chemical controls are available. For annual crops, a sensitive genotype can be replaced with disease-resistant varieties within a relatively short period of time. Diseases affecting fruit trees and vines may cause considerable losses through the gradual accumulation of newly affected trees over a number of years. This review describes how an important disease emerged, probably not from ingress of a new pathogen, but more likely from changes in cultural practices. It brings together information from a range of literary sources on the historical background of the emergence of xyloporosis and the linkage of this epidemic event with the history of citrus cultivation and with the natural history of Hop stunt viroid and other citrus viroids endemic to the Near East and the Mediterranean region.
Reichert and Perlberger reported on a “new” disease, xyloporosis, which appeared in 1928 among many of the newly planted citrus groves of Shamouti sweet orange trees that had been grafted onto Palestinian sweet lime , the most commonly used root stock in the British mandate-ruled area of Palestine. These authors described 3 essential phases of xyloporosis. First,wholesale plant containers small depressions appear on the stem bark of the root stock with small conoid pits with interfacing brownish pegs in the inner part of the bark. These symptoms often appear within 1 year of grafting and are most noticeable close to the bud union. In the second stage these symptoms intensify: The wood becomes discolored and, typically, the young tree becomes bent over and its leaves show symptoms typical of trees with root rot. In the third stage, there is blackish discoloration on the bark, the bark splits and leaves are small and yellow. Eventually, the branches wilt and die. Following intensive observations of a large number of infected trees of different ages in different planting areas, Reichert and Perlberger concluded that xyloporosis was present in all parts of the country and was not associated with any specific horticultural practice. Furthermore, although some differences were noticed among trees subject to different edaphic conditions, the disease could not be associated with any specific soil problem. The disease symptoms intensified as the trees aged. Seed source did not affect disease incidence. Pathological tests indicated that the disease was not associated with culturable fungal or bacterial pathogens. These observations led the authors to suggest 2 possible etiological causes of the disease: an unprecedented physiological disorder or an unknown viral pathogen. A major contribution to xyloporosis research was the observation of transmission by grafting of the cachexia disease affecting mandarins to a number of hosts, including a few that showed xyloporosis-like symptoms . These results suggested that the names of the 2 diseases, xyloporosis and cachexia, are synonymous and, based on the convention of prioritizing scientific names, the name cachexia was proposed for both diseases. Cachexia was found throughout most or all citrus-growing areas, particularly among Mediterranean varieties in Florida. Attempts by Norman and Childs to spread the cachexia disease via 5 different insect species failed and Olson showed that cachexia is not transmitted through seed. Later studies, reviewed by Bar-Joseph , showed that not only xyloporosis, but also exocortis and 3 other citrus viroids, were not seed transmitted to citron seedlings. The finding by Calavan and Christiansen that ‘Parsons Special’ mandarin showed more distinct symptoms than PSL supported the notion that the disease agents that cause xyloporosis and cachexia on different hosts are closely similar or identical. Recently, Langgut et al. reported the finding of typical citron pollen grains among the extracts from one layer of plaster, deposited to prevent leakage from an ancient irrigation pool belonging to a royal palace garden at present-day Kibbutz Ramat Rachel, near Jerusalem. Archeological evidence dated the construction of the facility to the Persian period and provides the first physical evidence for the earliest cultivation of citrus in the province of Judea. Initially, local growers refrained from grafting citron trees and were not familiar with the grafting technique .
The second edition of this book reported that some growers had started practicing grafting of citron on the PSL root stock, instead of propagation as seedling plants. Grafting was necessary because of the emergence of the destructive phytophthora gummosis disease, which rapidly decimated non-grafted trees and entire citrus industries throughout the Mediterranean basin . The dependence on grafting to save trees from diseases coincided with the selection of the Shamouti orange, which was obtained from a bud mutation of the local orange . Unlike the fruit of its seedy parent, the Shamouti fruit was almost seedless and these trees were propagated by grafting onto root stocks of an easily rooting citrus species, the PSL.Citrus cultivation expanded beyond the coastal plain , where the Shamouti orange on PSL root stocks was performing excellently, to new production areas just 10 km east of Jaffa, where all of the trees rapidly succumbed to the phytophthora gummosis disease. Phytophthora damage on PSL was initially blamed on the heavier soils of the new planting areas. However, when local citriculture expanded to sandy soils the root rot problems continued despite the improved edaphic conditions. It took almost 50 years for researchers and growers to realize that PSL root stocks made from cuttings were different from juvenile PSL seedlings, which were more sensitive to phytophthora root rot, and that the source of this difference was the absence in PSL seedlings of the viroid load commonly present in the root stocks that were made from cuttings. The positive effect of citrus viroid infection, the acquired resistance of phytophthora-sensitive root stocks induced by viroid infection, was first noticed by Rossetti et al. in Brazil. They noted that trees grafted onto Rangpur lime seedlings were succumbing to gummosis while those infected by the citrus exocortis viroid remained unaffected. Later studies using viroid-free and viroid infested buds of Shamouti grafted on PSL and also of other citrus stionic combinations confirmed this observation. Solel et al. showed that viroid infection also provides citron and Rangpur lime with tolerance to another serious fungal disease, mal secco . In retrospect, the change in susceptibility to phytophthora root rot could now be associated with the shift from the traditional practice of raising PSL root stocks from cuttings to producing root stocks from seed.