Dissection of transcriptional events in graft incompatible reactions of "Bearss" lemon (Citrus limon) and "Valencia" sweet orange (C. sinensis) on a novel citrandarin (C. reticulata × Poncirus trifoliata) rootstock.

Citrus is commercially propagated via grafting, which ensures trees have consistent fruit traits combined with favorable traits from the rootstock such as soil adaptability, vigor, and resistance to soil pathogens. Graft incompatibility can occur when the scion and rootstock are not able to form a permanent, healthy union. Understanding and preventing graft incompatibility is of great importance in the breeding of new fruit cultivars and in the choice of scion and rootstock by growers. The rootstock US-1283, a citrandarin generated from a cross of "Ninkat" mandarin (Citrus reticulata) and "Gotha Road" #6 trifoliate orange (Poncirus trifoliata), was released after years of field evaluation because of its superior productivity and good fruit quality on "Hamlin" sweet orange (C. sinensis) under Florida's growing conditions. Subsequently, it was observed that trees of "Bearss" lemon (C. limon) and "Valencia" sweet orange (C. sinensis) grafted onto US-1283 exhibited unhealthy growth near the graft union. The incompatibility manifested as stem grooving and necrosis underneath the bark on the rootstock side of the graft. Another citrandarin rootstock, US-812 (C. reticulata "Sunki" × P. trifoliata "Benecke"), is fully graft compatible with the same scions. Transcriptome analysis was performed on the vascular tissues above and below the graft union of US-812 and US-1283 graft combinations with "Bearss" and "Valencia" to identify expression networks associated with incompatibility and help understand the processes and potential causes of incompatibility. Transcriptional reprogramming was stronger in the incompatible rootstock than in the grafted scions. Differentially expressed genes (DEGs) in US-1283, but not the scions, were associated with oxidative stress and plant defense, among others, similar to a pathogen-induced immune response localized to the rootstock; however, no pathogen infection was detected. Therefore, it is hypothesized that this response could have been triggered by signaling miscommunications between rootstock and scion either through (1) unknown molecules from the scion that were perceived as danger signals by the rootstock, (2) missing signals from the scion or missing receptors in the rootstock necessary for the formation of a healthy graft union, (3) the overall perception of the scion by the rootstock as non-self, or (4) a combination of the above.

A Chronological Study on Grapevine Leafroll-Associated Virus 2 in Australia.

Grapevine leafroll disease affects the health status of grapevines worldwide. Most studies in Australia have focused on grapevine leafroll-associated viruses 1 and 3, while little attention has been given to other leafroll virus types, in particular, grapevine leafroll-associated virus 2 (GLRaV-2). A chronological record of the temporal occurrence of GLRaV-2 in Australia since 2001 is reported. From a total of 11,257 samples, 313 tested positive, with an overall incidence of 2.7%. This virus has been detected in 18 grapevine varieties and Vitis rootstocks in different regions of Australia. Most varieties were symptomless on their own roots, while Chardonnay showed a decline in virus-sensitive rootstocks. An isolate of GLRaV-2, on own-rooted Vitis vinifera cv. Grenache, clone SA137, was associated with severe leafroll symptoms after veraison with abnormal leaf necrosis. The metagenomic sequencing results of the virus in two plants of this variety confirmed the presence of GLRaV-2, as well as two inert viruses, grapevine rupestris stem pitting-associated virus (GRSPaV) and grapevine rupestris vein feathering virus (GRVFV). No other leafroll-associated viruses were detected. Among the viroids, hop stunt viroid and grapevine yellow speckle viroid 1 were detected. Of the six phylogenetic groups identified in GLRaV-2, we report the presence of four groups in Australia. Three of these groups were detected in two plants of cv. Grenache, without finding any recombination event. The hypersensitive reaction of certain American hybrid rootstocks to GLRaV-2 is discussed. Due to the association of GLRaV-2 with graft incompatibility and vine decline, the risk from this virus in regions where hybrid Vitis rootstocks are used cannot be overlooked.

Concerning the Etiology of Syrah Decline: A Fresh Perspective on an Old and Complex Issue Facing the Global Grape and Wine Industry.

Syrah decline, first identified in Southern France in the 1990s, has become a major concern in the global grape and wine industry. This disease mainly affects Syrah (Shiraz) grapevines. Characteristic symptoms include the bright and uniform reddening of leaves throughout the canopy in late summer or early fall; the appearance of abnormalities on the trunk, mainly at the graft union (swelling, pits, grooves, and necrosis); and a reduction in vine vigor, yield and berry quality. Diseased vines may die a few years after disease onset. Damages to the vine are even more pronounced in cool climate regions such as Ontario (Canada), where the affected vines are subjected to very cold and prolonged winters, leading to large numbers of vine deaths. Despite the extensive efforts of the global grape research community over the past few decades, the etiology of this disease remains unclear. In this study, we conducted extensive analyses of viruses in declining Syrah vines identified in commercial vineyards in the Niagara region (Ontario, Canada) through high-throughput sequencing, PCR, RT-PCR and the profiling of genetic variants of select viruses. Multiple viruses and viral strains, as well as three viroids, were identified. However, an unequivocal causal relationship cannot be established between Syrah decline and any of these viruses, although the possibility that certain virus or genetic variants, or both in combination, may contribute to the disease cannot be excluded. Gleaning all information that is available to date, we feel that the traditional approach and an insistence on finding a single cause for such a complex disorder in a woody perennial fruit crop involving grafting will prove to be futile. We hope that this study offers new conceptual perspectives on the etiology of this economically important but enigmatic disease complex that affects the global grape and wine industry.

Metabolite profiling and transcriptome analyses provide insight into the regulatory network of graft incompatibility in litchi.

Litchi is an important commercial fruit crop widely grown in the world. Graft incompatibility between rootstocks and scions is a major constraint for large-scale cultivation of litchi orchards, popularization of new and excellent litchi varieties, and associated industrial development. Further, the genetic mechanism of graft incompatibility is still unclear in litchi. To reduce the incompatibility problems, this study investigated metabolic and transcriptomic differences between graft compatible and incompatible rootstock-scion combinations of litchi. The result of metabolomics analysis showed that incompatible rootstock-scion interaction modified the profiles of several metabolic substances. However, various compounds of flavonoids, phenolic acids, and lignin predominantly exhibited significantly altered abundance in graft incompatible combinations. Transcriptome analysis identified that graft incompatibility induces dynamic gene differences. The majority of these differentially expressed genes were enriched in biosynthetic pathways of phenylpropanoids. The differential expressions of genes in these pathways could be linked to the differential abundance levels of flavonoids, phenolic acids, and lignin compounds. Integrated metabolomic and transcriptomic analyses revealed a strong relationship between differential genes and differential metabolites identified in this study. In addition, identified hub genes and metabolites were closely associated with graft incompatibility of litchi. This study characterized the abundance of metabolites and genes in graft incompatible combinations and further discussed the genetic mechanism of graft incompatibility in litchi. Our results provide a platform to dissect the molecular mechanisms of graft incompatibility in the litchi fruit.

Rootstock identity in melon-pumpkin graft combinations determines fruit metabolite profile.

Grafting has the potential to improve melon fruit yield and quality, but it is currently held that a lack of compatibility between the rootstock and scion compromises such an effect. To throw light on this subject, we studied melon-pumpkin graft combinations with different levels of compatibility to assess to the effect of the rootstock identity on melon fruit yield and quality, including total fruit ortho-diphenols, total flavonoids, and primary fruit metabolites. Melon cv. 'Kiran' (Ki) was grafted onto three pumpkin rootstocks, 'TZ-148' (TZ), 'Shimshon' (Sh), and '53006' (r53), characterized by high, moderate, and low compatibility, respectively. The non-grafted melon cultivar Ki was used as the control. The incompatible combination Ki/r53 gave the lowest fruit yield and the lowest average fruit weight. In that combination, the content of total ortho-diphenols increased vs. Ki and Ki/TZ and that of total flavonoids decreased vs. Ki/Sh. In addition, concentrations of the amino acids, glutamate, methionine, valine, alanine, glycine, and serine, increased in the pulp of the two compatible combinations, i.e., Ki/TZ and Ki/Sh, suggesting that rootstock identity and compatibility with melon Ki scion modulated amino acid synthesis. Our results show an association between rootstock identity (and level of compatibility with the scion) and an enhancement of fruit nutritional values, i.e., high concentrations of organic acids (determined as citrate, malate, fumarate, and succinate) and soluble carbohydrates (sucrose) were recorded in the pulp of the two compatible combinations, i.e., Ki/TZ and Ki/Sh.

Mapping Quantitative Trait Loci Associated With Graft (In)Compatibility in Apricot (Prunus armeniaca L.).

Graft incompatibility (GI) between the most popular Prunus rootstocks and apricot cultivars is one of the major problems for rootstock usage and improvement. Failure in producing long-leaving healthy grafts greatly affects the range of available Prunus rootstocks for apricot cultivation. Despite recent advances related to the molecular mechanisms of a graft-union formation between rootstock and scion, information on genetic control of this trait in woody plants is essentially missing because of a lack of hybrid crosses, segregating for the trait. In this study, we have employed the next-generation sequencing technology to generate the single-nucleotide polymorphism (SNP) markers and construct parental linkage maps for an apricot F1 population "Moniqui (Mo)" × "Paviot (Pa)" segregating for ability to form successful grafts with universal Prunus rootstock "Marianna 2624". To localize genomic regions associated with this trait, we genotyped 138 individuals from the "Mo × Pa" cross and constructed medium-saturated genetic maps. The female "Mo" and male "Pa" maps were composed of 557 and 501 SNPs and organized in eight linkage groups that covered 780.2 and 690.4 cM of genetic distance, respectively. Parental maps were aligned to the Prunus persica v2.0 genome and revealed a high colinearity with the Prunus reference map. Two-year phenotypic data for characters associated with unsuccessful grafting such as necrotic line (NL), bark and wood discontinuities (BD and WD), and an overall estimate of graft (in)compatibility (GI) were collected for mapping quantitative trait loci (QTLs) on both parental maps. On the map of the graft-compatible parent "Pa", two genomic regions on LG5 (44.9-60.8 cM) and LG8 (33.2-39.2 cM) were associated with graft (in)compatibility characters at different significance level, depending on phenotypic dataset. Of these, the LG8 QTL interval was most consistent between the years and supported by two significant and two putative QTLs. To our best knowledge, this is the first report on QTLs for graft (in)compatibility in woody plants. Results of this work will provide a valuable genomic resource for apricot breeding programs and facilitate future efforts focused on candidate genes discovery for graft (in)compatibility in apricot and other Prunus species.

Identifying Molecular Markers of Successful Graft Union Formation and Compatibility.

Grafting is a technique used for millennia for vegetative propagation, especially in perennial fruit crops. This method, used on woody and herbaceous plants, can improve several agronomic characteristics, such as yield or vigor, as well as tolerance to biotic and abiotic stresses. However, some scion/rootstock combinations suffer from poor graft compatibility, i.e., they are unable to form and/or sustain a successful graft union. Identifying symptoms of graft incompatibility is difficult because they are not always present in the first years after grafting and in most cases the causes of incompatibility are still poorly understood. Studies of changes in transcript abundance during graft union formation indicate that grafting responses are similar to responses to wounding and include the differential expression of genes related to hormone signaling, oxidative stress, formation of new vascular vessels, cell development, and secondary metabolites, in particular polyphenols. This review summarizes current knowledge of the changes in transcript abundance, redox status and metabolites accumulation during graft union formation and in cases of graft incompatibility. The goal of this review is to discuss the possibility of identifying marker transcripts, enzyme activities and/or metabolites of grafting success and graft compatibility which could be used to score grafting success for genetic research and in breeding programs. We highlight gaps in current knowledge and potential research directions in this field.

Living with Two Genomes: Grafting and Its Implications for Plant Genome-to-Genome Interactions, Phenotypic Variation, and Evolution.

Plant genomes interact when genetically distinct individuals join, or are joined, together. Individuals can fuse in three contexts: artificial grafts, natural grafts, and host-parasite interactions. Artificial grafts have been studied for decades and are important platforms for studying the movement of RNA, DNA, and protein. Yet several mysteries about artificial grafts remain, including the factors that contribute to graft incompatibility, the prevalence of genetic and epigenetic modifications caused by exchanges between graft partners, and the long-term effects of these modifications on phenotype. Host-parasite interactions also lead to the exchange of materials, and RNA exchange actively contributes to an ongoing arms race between parasite virulence and host resistance. Little is known about natural grafts except that they can be frequent and may provide opportunities for evolutionary innovation through genome exchange. In this review, we survey our current understanding about these three mechanisms of contact, the genomic interactions that result, and the potential evolutionary implications.

Malus pumila 'Spy 227' and Apple stem pitting virus: graft incompatibility and epinasty.

Apple stem pitting foveavirus (ASPV) is one of the most important and widespread virus infecting apples in the world. Of late, the virus has been found to be invariably associated with most of the apple plantations of Shimla district of Himachal Pradesh based on DAS-ELISA results. Bioassay of viruses in vegetatively propagated crops including apple is considered to be an essential component in indexing programmes for the production of virus free propagating material. Woody indicator Malus pumila 'Spy 227' was used for the detection of ASPV through double grafting method. Graft incompatibility and epinasty symptoms were observed on Malus pumila Spy 227 indicator plants. Further, molecular identification of the virus isolate was done by cloning and sequencing of the test isolate. Partial sequence analysis of the coat protein gene showed 89 % nucleotide identity in BLASTN analysis with ASPV isolate from China (Accession No. JF895517). This is the first record of ASPV producing Graft incompatibility on Spy 227 indicator plants.

Incompatibilidade de enxertia induz aumento da suscetibilidade de cultivares de pessegueiro à Xanthomonas arboricola pv. Pruni / Graft incompatibility induces susceptibility increase of peach cultivars to Xanthomonas arboricola pv. Pruni

O objetivo deste trabalho foi avaliar a compatibilidade de enxertia, a suscetibilidade à bacteriose e a frutificação de oito combinações, assim como, a influência da compatibilidade sobre a severidade da doença e a frutificação do pessegueiro. O experimento foi realizado ao nível de campo, sob infecção natural da bactéria, no Centro Agropecuário da Palma, pertencente à FAEM/UFPel, em Capão do Leão-RS. Foram avaliadas as combinações entre as cultivares 'Chimarrita' e Maciel e os porta-enxertos 'Aldrighi', 'Capdeboscq', 'Tsukuba 1' e 'Umezeiro'. O pomar foi implantado em delineamento experimental de blocos casualizados com quatro repetições, sendo cada unidade experimental composta de cinco plantas. Foi avaliada, a compatibilidade de enxertia, o diâmetro do caule, a severidade de dano e o índice de doença, a área foliar e a frutificação. As combinações entre as cultivares 'Chimarrita' e 'Maciel' e o porta-enxerto 'Umezeiro', apresentaram menor compatibilidade de enxertia, maior severidade de dano e índice de bacteriose, além de menor frutificação que as demais. Os resultados obtidos possibilitaram concluir que as combinações 'Chimarrita'/'Umezeiro' e 'Maciel'/'Umezeiro' são incompatíveis, além de, mais suscetíveis à bacteriose e menos produtivas, comparadas às demais. Pode-se dizer também, que a incompatibilidade de enxertia induz maior suscetibilidade das plantas à infecção por bacteriose, resultando em maior severidade da doença e menor frutificação.

The objective of this study was to evaluate the graft compatibility, susceptibility to bacterial leaf spot and fruiting on eight combinations, as well as the influence of graft compatibility in the severity of the disease and fruiting. The research was conducted at the field under natural bacterial infection, in the Agricultural Center of Palma, belonging to the FAEM/UFPel, in Capão do Leão-RS. It was evaluated the combinations composed by grafting among the 'Chimarrita' and 'Maciel' cultivars and the rootstocks 'Aldrighi', 'Capdeboscq', 'Tsukuba 1' and 'Umezeiro'. The orchard was implanted in a randomized block design with four replications, each experimental unit composed of five plants. The graft compatibility, stem diameter, injury severity, disease index, leaf area and fruiting were evaluated. The combinations between 'Chimarrita' and 'Maciel' cultivars and 'Umezeiro' rootstocks showed lower graft compatibility, highest injury severity, disease index, as well as, lower fruiting. The results obtained allow to conclude that 'Chimarrita'/'Umezeiro' and 'Maciel'/ 'Umezeiro' combinations are incompatibles, more susceptible to bacterial leaf spot and less productive compared to the others. Furthermore, the graft incompatibility induces higher susceptibility of plants to bacterial leaf spot infection, resulting in greater disease severity and lower fruiting.

Phenolic compounds involved in grafting incompatibility of Vitis spp: development and validation of an analytical method for their quantification.

INTRODUCTION: Graft incompatibility of Vitis spp is an unresolved worldwide problem with important economic consequences. Grafting comprises a complex set of morphological and physiological alterations, in which the phenolic compounds seem to be strongly involved. Therefore, a detailed analysis and recognition of structural phenolic compounds diversity in the two partners of a Vitis graft is of great importance to evaluate their role as markers of graft establishment. OBJECTIVE: To optimise a sample extraction method, and to develop and validate a high-performance liquid chromatography (HPLC) method for the simultaneous determination of phenolic acids and flavonols in the graft union so as to understand their behaviour in the metabolism of the scion-rootstock system, using compatible and incompatible combinations of a Syrah cultivar and two rootstocks (R110 and SO4). METHODS: Sixty extracts of Vitis grafting tissues were prepared and analysed by HPLC for the qualitative and quantitative determination of their phenolic profile. RESULTS: Among the phenolic compounds identified in the samples, one benzoic acid (gallic acid), three cinnamic acids (caffeic acid, ferulic acid and sinapic acid) and two flavonols (catechin and epicatechin) are potentially suitable as markers of graft incompatibility. CONCLUSION: The method developed presents good performance and lends itself readily for application in routine analysis of the phenolic composition of Vitis grafting tissues to distinguish compatible and incompatible combinations in the graft callusing stage.

Somatic hybrid plantlets regeneration between Citrus and its wild relative, Murraya paniculata via protoplast electrofusion.

Protoplasts isolated from `Page' tangelo (Minneola tangelo × clementine) cell suspension cultures were electrically fused with mesophyll protoplasts of orange jessamine [Murraya paniculata (L.) Jack]. Shoots were regenerated after 6 - 10 months of culture, but they were extremely recalcitrant to producing roots in root-induction medium. Complete plantlets were formed via micrografting. Chromosome counting of shoot tips revealed they were tetraploids (2n = 4x = 36). Glutamateoxaloacetate transaminase isozyme and randomly amplified polymorphic DNA analysis confirmed their hybridity. Orange jessamine is immune to citrus huanglongbin, a severe disease of citrus, but sexual incompatibility and limited graft compatibility exist between Citrus and orange jessamine. The cell fusion technique may make it possible to transfer the huanglongbin resistance trait from orange jessamine to Citrus.