Biological and molecular characterization of a resistance-breaking isolate of citrus tristeza virus from Uruguay and its effects on Poncirus trifoliata growth performance.

Resistance-breaking (RB) isolates of citrus tristeza virus (CTV) can replicate and move systemically in Poncirus trifoliata, a rootstock widely used for management of decline caused by CTV and other purposes. In Uruguay, severe CTV isolates are prevalent, and an RB isolate (designated as RB-UY1) was identified. In order to predict the implications of this genotype circulating in citrus crops grafted on trifoliate rootstocks, the aim of this work was to determine the biological and molecular characteristics of this isolate, the efficiency of its transmission by Toxoptera citricida, and its effects on plant growth performance of P. trifoliata. Our results show that RB-UY1 can be classified as a mild isolate, that it is phylogenetically associated with the RB1 group, and that it is efficiently transmitted by T. citrida. They also suggest that the RB-UY1 isolate should not affect the performance of citrus crops grafted on trifoliate rootstocks, although some growth parameters of P. trifoliata seedlings were affected four years after inoculation.

Presence of leaves and use of IBA in adventitious rooting, chlorophyll fluorescence, and root ontogeny of 'Fepagro C 13' and 'Trifoliata' cuttings / Presença de folhas e uso de AIB no enraizamento adventício, fluorescência da clorofila e ontogenia radicular em estacas de 'Fepagro C 13' e 'Trifoliata'

Cutting is proposed as an alternative method for the propagation of citrus rootstocks to obtain clonal material especially in varieties with a low rate of polyembryony and possibly reduce the seedling formation period. The aim of this study was to examine the influence of leaves and the application of indolebutyric acid (IBA) on adventitious rooting and chlorophyll fluorescence as well as to characterize root ontogeny in 'Fepagro C 13' and 'Trifoliata' citrus rootstock cuttings. Semi-hardwood cuttings were collected in December 2018 and subjected to the following treatments: presence or absence of leaves, with or without application of IBA (4000 mg L-1). Over 92 days in a greenhouse environment, leaf abscission percentage, cutting mortality, rooting percentage, and number of roots per cutting were evaluated. Additionally, initial (Fo), maximum (Fm), and variable (Fv) chlorophyll fluorescence, photochemical efficiency (Fv/Fm), and Fv/Fo ratio were analyzed. Tissue samples from the base of the cutting were collected for the anatomical study. The presence of leaves on 'Fepagro C 13' and 'Trifoliata' cuttings is essential for adventitious rooting. The use of auxin influences the photochemical efficiency of leaves on the cuttings. In 'Trifoliata' cuttings, adventitious roots originate from the vascular cambium.(AU)

A estaquia é proposta como método alternativo para a propagação de porta-enxertos de citros, visando a garantia de obtenção de material clonal, especialmente em variedades com baixa taxa poliembriônica, e uma possível redução no período para a obtenção das mudas. O objetivo deste estudo foi avaliar a influência das folhas e da aplicação de ácido indolbutírico (AIB) sobre o enraizamento adventício, fluorescência da clorofila e caracterizar a ontogenia radicular em estacas dos porta-enxertos citrangeiro 'Fepagro C 13', e 'Trifoliata'. Estacas semilenhosas foram coletadas em dezembro de 2018 e submetidas aos tratamentos: presença ou ausência de folhas com aplicação ou não de AIB (4000 mg L-1). Ao longo de 92 dias em casa de vegetação foram avaliados os percentuais de abscisão foliar, mortalidade de estacas, percentual de enraizamento e número de raízes por estaca. Também os dados da fluorescência inicial (Fo), máxima (Fm) e variável (Fv) da clorofila, a eficiência fotoquímica (Fv/Fm) e a razão Fv/Fo foram avaliados. Amostras de tecido da base da estaca foram coletadas para a realização do estudo anatômico. A presença das folhas nas estacas de 'Fepagro C 13' e 'Trifoliata' é essencial para o enraizamento adventício. O uso de auxina influencia a eficiência fotoquímica nas folhas das estacas. Em estacas de 'Trifoliata' as raízes adventícias originam-se a partir do câmbio vascular.(AU)

Wide-ranging transcriptomic analysis of Poncirus trifoliata, Citrus sunki, Citrus sinensis and contrasting hybrids reveals HLB tolerance mechanisms.

Huanglongbing (HLB), caused mainly by 'Candidatus Liberibacter asiaticus' (CLas), is the most devastating citrus disease because all commercial species are susceptible. HLB tolerance has been observed in Poncirus trifoliata and their hybrids. A wide-ranging transcriptomic analysis using contrasting genotypes regarding HLB severity was performed to identify the genetic mechanism associated with tolerance to HLB. The genotypes included Citrus sinensis, Citrus sunki, Poncirus trifoliata and three distinct groups of hybrids obtained from crosses between C. sunki and P. trifoliata. According to bacterial titer and symptomatology studies, the hybrids were clustered as susceptible, tolerant and resistant to HLB. In P. trifoliata and resistant hybrids, genes related to specific pathways were differentially expressed, in contrast to C. sinensis, C. sunki and susceptible hybrids, where several pathways were reprogrammed in response to CLas. Notably, a genetic tolerance mechanism was associated with the downregulation of gibberellin (GA) synthesis and the induction of cell wall strengthening. These defense mechanisms were triggered by a class of receptor-related genes and the induction of WRKY transcription factors. These results led us to build a hypothetical model to understand the genetic mechanisms involved in HLB tolerance that can be used as target guidance to develop citrus varieties or rootstocks with potential resistance to HLB.

Establishment and optimization of a hydroponic system for root morphological and nutritional analysis of citrus

The hydroponic growth system is a convenient platform for studying whole plant physiology, especially for root morphological and nutritional analysis. However, we found that most hydroponic systems described in the literature are not suitable for citrus plants. In this study, a hydroponic system for citrus was designed, comprising three principal components: power and time switch, aeration and hydroponic culture. Herein, details of the protocol were described, including equipment setup, seed pregermination and cultivation, together with preparation and transfer of nutrient solution into hydroponics. In order to demonstrate the adaptability of the trifoliate orange plant to our hydroponic system, comparative tests between soil- and hydroponically-grown plants were carried out. The results showed that the plants grew normally and there were no obvious differences between soil- and hydroponically-grown plants. In addition, nutrient deficiency and transcriptional analysis were carried out to test the efficiency, functionality and suitability of our hydroponic system for the application of physiological and molecular analysis. The results, compared with previous studies, showed that our hydroponic system delivered superior performance as regards the physiological and molecular analysis. Taken together, we established the culture system which is best suited for the growth of trifoliate oranges under hydroponic conditions. The hydroponic system described in this paper is easily constructed and controlled at a low cost. It may serve a wide gamut of experimental purposes, especially root morphological and nutritional analysis of trifoliate oranges and the system is also adaptable to other citrus plants by varying the device size.(AU)

Establishment and optimization of a hydroponic system for root morphological and nutritional analysis of citrus

The hydroponic growth system is a convenient platform for studying whole plant physiology, especially for root morphological and nutritional analysis. However, we found that most hydroponic systems described in the literature are not suitable for citrus plants. In this study, a hydroponic system for citrus was designed, comprising three principal components: power and time switch, aeration and hydroponic culture. Herein, details of the protocol were described, including equipment setup, seed pregermination and cultivation, together with preparation and transfer of nutrient solution into hydroponics. In order to demonstrate the adaptability of the trifoliate orange plant to our hydroponic system, comparative tests between soil- and hydroponically-grown plants were carried out. The results showed that the plants grew normally and there were no obvious differences between soil- and hydroponically-grown plants. In addition, nutrient deficiency and transcriptional analysis were carried out to test the efficiency, functionality and suitability of our hydroponic system for the application of physiological and molecular analysis. The results, compared with previous studies, showed that our hydroponic system delivered superior performance as regards the physiological and molecular analysis. Taken together, we established the culture system which is best suited for the growth of trifoliate oranges under hydroponic conditions. The hydroponic system described in this paper is easily constructed and controlled at a low cost. It may serve a wide gamut of experimental purposes, especially root morphological and nutritional analysis of trifoliate oranges and the system is also adaptable to other citrus plants by varying the device size.

PpCRN7 and PpCRN20 of Phythophthora parasitica regulate plant cell death leading to enhancement of host susceptibility.

BACKGROUND: Phytophthora species secrete cytoplasmic effectors from a family named Crinkler (CRN), which are characterised by the presence of conserved specific domains in the N- and C-terminal regions. P. parasitica causes disease in a wide range of host plants, however the role of CRN effectors in these interactions remains unclear. Here, we aimed to: (i) identify candidate CRN encoding genes in P. parasitica genomes; (ii) evaluate the transcriptional expression of PpCRN (Phytophthora parasitica Crinkler candidate) during the P. parasitica interaction with Citrus sunki (high susceptible) and Poncirus trifoliata (resistant); and (iii) functionally characterize two PpCRNs in the model plant Nicotiana benthamiana. RESULTS: Our in silico analyses identified 80 putative PpCRN effectors in the genome of P. parasitica isolate 'IAC 01/95.1'. Transcriptional analysis revealed differential gene expression of 20 PpCRN candidates during the interaction with the susceptible Citrus sunki and the resistant Poncirus trifoliata. We have also found that P. parasitica is able to recognize different citrus hosts and accordingly modulates PpCRNs expression. Additionally, two PpCRN effectors, namely PpCRN7 and PpCRN20, were further characterized via transient gene expression in N. benthamiana leaves. The elicitin INF-1-induced Hypersensitivity Response (HR) was increased by an additive effect driven by PpCRN7 expression, whereas PpCRN20 expression suppressed HR response in N. benthamiana leaves. Despite contrasting functions related to HR, both effectors increased the susceptibility of plants to P. parasitica. CONCLUSIONS: PpCRN7 and PpCRN20 have the ability to increase P. parasitica pathogenicity and may play important roles at different stages of infection. These PpCRN-associated mechanisms are now targets of biotechnological studies aiming to break pathogen's virulence and to promote plant resistance.

Vigor and mortality of citrus progenies with potential use as rootstocks / 10.1590/1678-992x-2016-0455

Rootstocks influence certain characteristics of the scion, such as vigor, fruit yield and quality, and tolerance to biotic and abiotic factors. In conventional citrus breeding programs, hybridization is the main method used for obtaining rootstock varieties. Between Sept and Oct 2006, controlled pollinations were carried out resulting in nine progenies, totaling 461 hybrid seedlings. The female parents were the ‘Santa Cruz Rangpur lime (SCRL) and the ‘Sunki of Florida mandarin (SFM), while the male parents, in addition to this mandarin, were the ‘Indio and ‘Riverside citrandarins, the ‘Thomasville citrangequat (THOM), the ‘Swingle citrumelo, the ‘Argentina citrange (AGC) and the ‘Benecke trifoliate orange (BKTO). Fruit setting rates of these crosses were measured and the hybrids generated were planted in the field in 2008 and evaluated from 2010 to 2014, for the variables of plant mortality, visual vigor, plant height and trunk diameter. The descriptive statistics mean, variation range, coefficient of variation and Pearson skewness coefficient were calculated. Data were submitted to analysis of variance and the means of the treatments were grouped by the Scott-Knott test, both at 5 % significance. The progenies, having the ‘Sunki mandarin as the female parent, presented higher fruit setting rates and lower plant mortality rates than those obtained from the female parent ‘Rangpur lime, while zero plant mortality was observed in the SFM × BKTO cross. The crosses SCRL × SFM, SFM × BKTO, SFM × AGC and SFM × THOM have the potential to generate vigorous hybrids, resulting in rootstock varieties adapted to the landscape unit of Coastal Tableland.(AU)

Molecular Basis of Citrus sunki Susceptibility and Poncirus trifoliata Resistance Upon Phytophthora parasitica Attack.

Coevolution has shaped the molecular basis of an extensive number of defense mechanisms in plant-pathogen interactions. Phytophthora parasitica, a hemibiothrophic oomycete pathogen and the causal agent of citrus root rot and gummosis, interacts differently with Citrus sunki and Poncirus trifoliata, two commonly favored citrus rootstocks that are recognized as susceptible and resistant, respectively, to P. parasitica. The molecular core of these interactions remains elusive. Here, we provide evidence on the defense strategies employed by both susceptible and resistant citrus rootstocks, in parallel with P. parasitica deployment of effectors. Time course expression analysis (quantitative real-time polymerase chain reaction) of several defense-related genes were evaluated during i) plant disease development, ii) necrosis, and iii) pathogen effector gene expression. In C. sunki, P. parasitica deploys effectors, including elicitins, NPP1 (necrosis-inducing Phytophthora protein 1), CBEL (cellulose-binding elicitor and lectin activity), RxLR, and CRN (crinkler), and, consequently, this susceptible plant activates its main defense signaling pathways that result in the hypersensitive response and necrosis. Despite the strong plant-defense response, it fails to withstand P. parasitica invasion, confirming its hemibiothrophic lifestyle. In Poncirus trifoliata, the effectors were strongly expressed, nevertheless failing to induce any immunity manipulation and disease development, suggesting a nonhost resistance type, in which the plant relies on preformed biochemical and anatomical barriers.

Vigor and mortality of citrus progenies with potential use as rootstocks / 10.1590/1678-992x-2016-0455

Rootstocks influence certain characteristics of the scion, such as vigor, fruit yield and quality, and tolerance to biotic and abiotic factors. In conventional citrus breeding programs, hybridization is the main method used for obtaining rootstock varieties. Between Sept and Oct 2006, controlled pollinations were carried out resulting in nine progenies, totaling 461 hybrid seedlings. The female parents were the ‘Santa Cruz Rangpur’ lime (SCRL) and the ‘Sunki of Florida’ mandarin (SFM), while the male parents, in addition to this mandarin, were the ‘Indio’ and ‘Riverside’ citrandarins, the ‘Thomasville’ citrangequat (THOM), the ‘Swingle’ citrumelo, the ‘Argentina’ citrange (AGC) and the ‘Benecke’ trifoliate orange (BKTO). Fruit setting rates of these crosses were measured and the hybrids generated were planted in the field in 2008 and evaluated from 2010 to 2014, for the variables of plant mortality, visual vigor, plant height and trunk diameter. The descriptive statistics mean, variation range, coefficient of variation and Pearson skewness coefficient were calculated. Data were submitted to analysis of variance and the means of the treatments were grouped by the Scott-Knott test, both at 5 % significance. The progenies, having the ‘Sunki’ mandarin as the female parent, presented higher fruit setting rates and lower plant mortality rates than those obtained from the female parent ‘Rangpur’ lime, while zero plant mortality was observed in the SFM × BKTO cross. The crosses SCRL × SFM, SFM × BKTO, SFM × AGC and SFM × THOM have the potential to generate vigorous hybrids, resulting in rootstock varieties adapted to the landscape unit of Coastal Tableland.

Effects of grafting on key photosynthetic enzymes and gene expression in the citrus cultivar Huangguogan.

Grafting influences scion photosynthetic capacity and fruit quality. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which strongly affects photosynthetic rate, and Rubisco activase (RCA), which regulates Rubisco activity, are two key photosynthetic enzymes. However, little information is available regarding the effect of grafting on the concentration and expression of Rubisco and RCA in the citrus cultivar Huangguogan. The objective of this study was to investigate the effect of grafting Huangguogan plants onto trifoliate orange, tangerine, and orange on: 1) the concentration of Rubisco and RCA; 2) the mRNA levels of rbcL, rbcS, and rca; and 3) fruit quality. Overall, the results showed that when Huangguogan plants budded on tangerine and orange, they had better fruit quality, while on trifoliate orange they had higher Rubisco concentration. Tangerine and orange are probably the most suitable rootstocks for Huangguogan plants given the environmental conditions of Sichuan Province, China.

Chromosome homeologies and high variation in heterochromatin distribution between Citrus L. and Poncirus Raf. as evidenced by comparative cytogenetic mapping.

The genus Citrus is well-known for its economic importance and complex taxonomy. Only three to six Citrus taxa are considered true biological species, among them is Citrus medica, the citron. Previous studies based on chromomycin A3 (CMA)/4',6-diamidino-2-phenylindole staining showed that the citron has a homomorphic karyotype, consisting of four distinct chromosome types according to the CMA(+) heterochromatin distribution. Based on the pattern of CMA(+) bands and the hybridization of 25 bacterial artificial chromosomes (BACs) from a genomic library of Poncirus trifoliata, a closely related species to the genus Citrus, we were able to identify each chromosome pair and build a comparative cytogenetic map for C. medica. The data showed a high degree of sequence conservation between these genera, enabling heterologous hybridization of BACs, and the establishment of chromosomal homeologies. It was thus possible to visualize changes in the position of some BACs in relation to CMA(+) bands. Since no breakdown of synteny was observed between these species, expansions and contractions in repetitive DNA sequences seem to be the major driving force of chromosomal evolution since the separation of these two genera.

Global gene expression of Poncirus trifoliata, Citrus sunki and their hybrids under infection of Phytophthora parasitica.

BACKGROUND: Gummosis and root rot caused by Phytophthora are among the most economically important diseases in citrus. Four F1 resistant hybrids (Pool R), and four F1 susceptible hybrids (Pool S) to P. parasitica, were selected from a cross between susceptible Citrus sunki and resistant Poncirus trifoliata cv. Rubidoux. We investigated gene expression in pools of four resistant and four susceptible hybrids in comparison with their parents 48 hours after P. parasitica inoculation. We proposed that genes differentially expressed between resistant and susceptible parents and between their resistant and susceptible hybrids provide promising candidates for identifying transcripts involved in disease resistance. A microarray containing 62,876 UniGene transcripts selected from the CitEST database and prepared by NimbleGen Systems was used for analyzing global gene expression 48 hours after infection with P. parasitica. RESULTS: Three pairs of data comparisons (P. trifoliata/C. sunki, Pool R/C. sunki and Pool R/Pool S) were performed. With a filter of false-discovery rate less than 0.05 and fold change greater than 3.0, 21 UniGene transcripts common to the three pairwise comparative were found to be up-regulated, and 3 UniGene transcripts were down-regulated. Among them, our results indicated that the selected transcripts were probably involved in the whole process of plant defense responses to pathogen attack, including transcriptional regulation, signaling, activation of defense genes participating in HR, single dominant genes (R gene) such as TIR-NBS-LRR and RPS4 and switch of defense-related metabolism pathway. Differentially expressed genes were validated by RT-qPCR in susceptible and resistant plants and between inoculated and uninoculated control plants CONCLUSIONS: Twenty four UniGene transcripts were identified as candidate genes for Citrus response to P. parasitica. UniGene transcripts were likely to be involved in disease resistance, such as genes potentially involved in secondary metabolite synthesis, intracellular osmotic adjustment, signal transduction pathways of cell death, oxidative burst and defense gene expression. Furthermore, our microarray data suggest another type of resistance in Citrus-Phytophthora interaction conferred by single dominant genes (R gene) since we encountered two previously reported R genes (TIR-NBS-LRR and RPS4) upregulated in the resistant genotypes relative to susceptible. We identified 7 transcripts with homology in other plants but yet unclear functional characterization which are an interesting pool for further analyses and 3 transcripts where no significant similarity was found. This is the first microarray study addressing an evaluation of transcriptional changes in response to P. parasitica in Citrus.

Mapping the chromosomes of Poncirus trifoliata Raf. by BAC-FISH.

In spite of the importance of Citrus in agriculture and recent progress in genetic mapping and cytogenetics of this group, chromosome mapping of Citrus species is still limited to rDNA probes. In order to obtain a better chromosome characterization of one species from this group, CMA/DAPI double staining followed by in situ hybridization using 45S rDNA and 24 BACs (BAC-FISH) were used on Poncirus trifoliata. The BACs used were obtained from a genomic library of this species and were selected by membrane hybridization using genomic DNA. Four of them were isolated from the Citrus tristeza virus (Ctv) resistance gene region. The P. trifoliata karyotype is composed of two chromosome pairs with one terminal and one proximal CMA(+) band (B type chromosomes), four chromosome pairs with a single CMA(+) band (D type) and three chromosome pairs without bands (F type). In situ hybridization with 13 of the BACs gave single copy signals on seven chromosome pairs. At least one BAC was mapped on each arm of the two B chromosome pairs. Among the four D chromosome pairs, two were identified by BACs mapped on the long arms, one has a 45S rDNA site and the other had no signal. Six BACs allowed identification of the three F chromosome pairs, with one pair hybridizing with four BACs from the Ctv resistance gene region. In summary, all nine chromosome pairs could be differentiated, seven of them by BAC-FISH, while the other two chromosomes could be recognized by the CMA(+) band pattern and 45S rDNA sites. This first BAC-FISH map gives a general framework for comparative genome structure and evolutionary studies in Citrus and Poncirus, allowing the integration of genetic and physical maps when these BACs are included.

A simple chromosomal marker can reliably distinguishes Poncirus from Citrus species.

Several chromosome types have been recognized in Citrus and related genera by chromomycin A(3 )(CMA) banding patterns and fluorescent in situ hybridization (FISH). They can be used to characterize cultivars and species or as markers in hybridization and backcrossing experiments. In the present work, characterization of six cultivars of P. trifoliata ("Barnes", "Fawcett", "Flying Dragon", "Pomeroy", "Rubidoux", "USDA") and one P. trifoliata x C. limonia hybrid was performed by sequential analyses of CMA banding and FISH using 5S and 45S rDNA as probes. All six cultivars showed a similar CMA(+) banding pattern with the karyotype formula 4B + 8D + 6F. The capital letters indicate chromosomal types: B, a chromosome with one telomeric and one proximal band; D, with only one telomeric band; F, without bands. In situ hybridization labeling was also similar among cultivars. Three chromosome pairs displayed a closely linked set of 5S and 45S rDNA sites, two of them co-located with the proximal band of the B type chromosomes (B/5S-45S) and the third one co-located with the terminal band of a D pair (D/5S-45S). The B/5S-45S chromosome has never been found in any citrus accessions investigated so far. Therefore, this B chromosome can be used as a marker to recognize the intergeneric Poncirus x Citrus hybrids. The intergeneric hybrid analyzed here displayed the karyotype formula 4B + 8D + 6F, with two chromosome types B/5S-45S and two D/5S-45S. The karyotype formula and the presence of two B/5S-45S chromosomes clearly indicate that the plant investigated is a symmetric hybrid. It also demonstrates the suitability of karyotype analyses to differentiate zygotic embryos or somatic cell fusions involving trifoliate orange germplasm.

Controle químico do cancro cítrico em plantas jovens sob manejo convencional e orgânico / Quemical control of citrus canker in young plants managed in conventional and organic systems

O objetivo deste trabalho foi avaliar o desenvolvimento de porta-enxertos de Poncirus trifoliata Raf. e de enxertos de laranjeira "Valência" (Citrus sinensis Osb.), os índices de ataque de cancro cítrico, causado pela bactéria Xanthomonas citri pv. citri, artificialmente inoculada, e o controle dessa moléstia, com pulverizações cúpricas em sistemas de cultivo, convencional e orgânico, no Centro de Formação da EMATER, situado no município de Montenegro, Estado do Rio Grande do Sul. Foram testadas pulverizações cúpricas a cada 7, 14 e 21 dias, com concentrações de 0,15 e 0,30 por cento de cobre metálico, utilizando calda bordalesa no sistema orgânico e oxicloreto de cobre no sistema convencional. Foram avaliados o crescimento do diâmetro do caule das plantas, a produção de matéria seca da parte aérea dos porta-enxertos, o número de folhas com lesões de cancro cítrico e o número de lesões presentes por folha atacada. Verificou-se que em ambos os sistemas de cultivo, no convencional e no orgânico, o desenvolvimento dos porta-enxertos foi semelhante, mas os enxertos cresceram mais no sistema convencional. Com elevada presença de fontes de inóculo, os tratamentos cúpricos não controlaram o cancro cítrico nos porta-enxertos. Já nos enxertos, com baixa presença de fontes de inóculo, tanto a calda bordalesa como o oxicloreto de cobre controlaram a doença, com melhor resposta na concentração de 0,3 por cento de cobre metálico, aplicada em intervalos de 14 dias. Para melhorar o controle do cancro cítrico, com calda bordalesa, em sistemas orgânicos de cultivo, devem ser desenvolvidas medidas eficazes de controle da larva-minadora.