Nursery performance of potentially promising rootstocks for citriculture in the south of Brazil

ABSTRACT: Diversification of rootstock varieties, with consequent reduction in phytosanitary risks, has great importance to the sustainability of citrus cultivation in the south of Brazil. This study evaluated the performance of 42 rootstocks in the nursery phase to generate ‘Valencia Late’ sweet orange seedlings. Therefore, nucellar seedlings from the rootstocks under study were analyzed in relation to plant height, stem diameter, mortality rate and percentage of bud set after grafting. The experimental design was completely randomized with four replications of 20 sample units. Not adapted genotypes had high mortality rates, and the ones of both hybrids LCR x CTSW - 009 and LVK x LCR - 038 were 100% and 90%, respectively. Other 13 genotypes had mortality rates above 30%, and ‘Sunki’ mandarin was the female genitor of ten of them. ‘Swingle’ citrumelo was the rootstock with the highest development (plant height and stem diameter), associated with low mortality rate. In relation to the other rootstocks, the best results were the fast grafting diameter, associated with low mortality rate obtained by hybrids HTR - 053, LRF x (LCR x TR) - 005, CLEO x TRBN - 245, CLEO x TRSW - 287, and citrandarins ‘Indio’, ‘Riverside’ and ‘San Diego’, as well as Trifoliate orange and ‘Rangpur’ lime. In addition to Trifoliate orange, which is widely used in Rio Grande do Sul, these rootstocks have great potential in citriculture in the south of Brazil.

Tree Growth and Production of Rainfed Valencia Sweet Orange Grafted onto Trifoliate Orange Hybrid Rootstocks under Aw Climate

Brazil is the largest producer of sweet orange and its juice in the world. Extensive cultivated area is located under an Aw climate in the North–Northwest of the state of São Paulo and the Triângulo of Minas Gerais state, being subjected to severe drought events. Although 56% of the orchards are irrigated in these regions, there is a need for drought tolerant rootstocks as an alternative to traditional genotypes such as Rangpur lime and Volkamer lemon, which are susceptible to the endemic citrus sudden death disease (CSD). In this sense, the tree size and production of Valencia sweet orange grafted onto 23 rootstock genotypes were evaluated over a ten-year period in rainfed cultivation at 7.0 m × 3.0 m spacing. Most evaluated types resulted from the cross of Poncirus trifoliata with Citrus, but two interspecific hybrids of Citrus (Sunki mandarin × Rangpur lime hybrids), the Barnes trifoliate orange and a tetraploid selection of Swingle citrumelo were also tested. Tropical Sunki mandarin was used as the reference control. Those hybrids coming from the cross of Sunki × Flying Dragon induced large tree sizes to Valencia sweet orange as well as the other citrandarins, Tropical Sunki mandarin and the Sunki mandarin × Rangpur lime hybrids, whereas only the tetraploid Swingle citrumelo behaved as a dwarfing rootstock, decreasing the canopy volume by 77% compared to that induced by the most vigorous citrandarin 535. The citrandarins 543 and 602 and the citrange C38 induced the highest mean fruit production, 67.2 kg·tree−1, but they also caused pronounced alternate bearing and only the hybrid 543 led to a high production efficiency consistently. Graft incompatibility symptoms were not observed over the evaluation period, and the canopy shape of Valencia sweet orange was also influenced by the rootstocks tested. Two citrandarins and one citrange were selected as the most promising alternative rootstocks for Valencia sweet orange grown under an Aw climate, even though productivity would likely benefit from supplementary irrigation.

Metabolomics Analysis Reveals Drought Responses of Trifoliate Orange by Arbuscular Mycorrhizal Fungi With a Focus on Terpenoid Profile

Soil water deficit seriously affects crop production, and soil arbuscular mycorrhizal fungi (AMF) enhance drought tolerance in crops by unclear mechanisms. Our study aimed to analyze changes in non-targeted metabolomics in roots of trifoliate orange (Poncirus trifoliata) seedlings under well-watered and soil drought after inoculation with Rhizophagus intraradices, with a focus on terpenoid profile. Root mycorrhizal fungal colonization varied from 70% under soil drought to 85% under soil well-watered, and shoot and root biomass was increased by AMF inoculation, independent of soil water regimes. A total of 643 secondary metabolites in roots were examined, and 210 and 105 differential metabolites were regulated by mycorrhizal fungi under normal water and drought stress, along with 88 and 17 metabolites being up-and down-regulated under drought conditions, respectively. KEGG annotation analysis of differential metabolites showed 38 and 36 metabolic pathways by mycorrhizal inoculation under normal water and drought stress conditions, respectively. Among them, 33 metabolic pathways for mycorrhization under drought stress included purine metabolism, pyrimidine metabolism, alanine, aspartate and glutamate metabolism, etc. We also identified 10 terpenoid substances, namely albiflorin, artemisinin (−)-camphor, capsanthin, β-caryophyllene, limonin, phytol, roseoside, sweroside, and α-terpineol. AMF colonization triggered the decline of almost all differential terpenoids, except for β-caryophyllene, which was up-regulated by mycorrhizas under drought, suggesting potential increase in volatile organic compounds to initiate plant defense responses. This study provided an overview of AMF-induced metabolites and metabolic pathways in plants under drought, focusing on the terpenoid profile.

Effect of Grafting Dates on Success and Growth of Kumquat (Fortunella japonica Swingle) Sapling

An experiment was carried out to study the effect of grafting dates on the success and growth of kumquat sapling during 2017-18 at Banepa, Kavre to standardize suitable grafting time. The experiment was conducted in a single factor completely randomized design with five treatments and three replications. The treatments consisted of five grafting dates which viz., 25th November, 10th December, 25th December, 10th January and 25th January. Scion collected from the mother plant of kumquat was grafted onto two years old trifoliate orange rootstock by side veneer method at 15 days intervals under screen house. The data were recorded at every 30 days interval and found a significant effect of grafting dates on most of the economically important parameters such as graft success percent, the mortality of sprouted grafts, sapling height, number of leaves per sapling and leaf area. The result revealed that 100% graft success was observed on the grafts prepared in January. The highest mortality (13.68±0.58%) was reported on 25th November grafted sapling. The sapling height (39.68±3.68 cm), number of leaves per sapling (23.12±3.43) and leaf area (16.34±1.05 cm2) were found superior for 10th January grafted sapling while the number of primary and secondary branches per sapling were found non-significant for all grafting dates. Therefore, the study revealed that 10th January was the most suitable time for grafting of kumquat under Kavre, Nepal conditions.

Exogenous Glomalin-Related Soil Proteins Differentially Regulate Soil Properties in Trifoliate Orange

Glomalin-related soil protein (GRSP) is a specific glycoprotein secreted into the soil by hyphae and spores of arbuscular mycorrhizal fungi that have many potential functions. It is not clear whether exogenous GRSP has an effect on plant growth and soil properties or whether the effects are related to the type of GRSP used. In this study, trifoliate orange (Poncirus trifoliata L. Raf.) seedlings were used to analyze the effects of easily extractable GRSP (EE-GRSP) and difficultly extractable GRSP (DE-GRSP) at a quarter-, half-, and full-strength concentration on shoot and root biomass as well as soil properties The results showed that, at different strengths, exogenous EE-GRSR significantly increased shoot and root biomass compared to the control, which displayed the most significant effects from the half-strength EE-GRSP. In contrast, DE-GRSP, at various strengths, significantly reduced shoot and root biomass. Furthermore, the application of exogenous EE-GRSP stimulated soil water-stable aggregate (WSA) content at 2–4 mm and 0.5–1 mm sizes, while DE-GRSP strongly reduced WSA content at the 2–4 mm, 1–2 mm, 0.5–1 mm, and 0.25–0.5 mm sizes, consequently leading to an increase or decrease in the WSA stability, according to the mean weight diameter. However, exogenous EE-GRSP decreased soil pH and DE-GRSP increased it, which was related to WSA stability. Exogenous EE-GRSP almost significantly increased soil acidic, neutral, and alkaline phosphatase activity at different strengths, while exogenous DE-GRSP, also at different strengths, significantly inhibited soil acidic phosphatase activity. The application of both exogenous EE-GRSP and DE-GRSP increased the organic carbon content of the soil. This study concluded that exogenous GRSP exerted differential effects on plant biomass and soil properties, and EE-GRSP can be considered as a soil stimulant for use in citrus plants. To our knowledge, this is the first report on the negative effects of exogenous DE-GRSP on plant biomass and soil properties.

Differential Effects of Exogenous Glomalin-Related Soil Proteins on Plant Growth of Trifoliate Orange Through Regulating Auxin Changes

Multiple functions of glomalin released by arbuscular mycorrhizal fungi are well-recognized, whereas the role of exogenous glomalins including easily extractable glomalin-related soil protein (EE-GRSP) and difficultly extractable glomalin-related soil protein (DE-GRSP) is unexplored for plant responses. Our study was carried out to assess the effects of exogenous EE-GRSP and DE-GRSP at varying strengths on plant growth and chlorophyll concentration of trifoliate orange (Poncirus trifoliata) seedlings, along with changes in root nutrient acquisition, auxin content, auxin-related enzyme and transporter protein gene expression, and element contents of purified GRSP. Sixteen weeks later, exogenous GRSP displayed differential effects on plant growth (height, stem diameter, leaf number, and biomass production): the increase by EE-GRSP and the decrease by DE-GRSP. The best positive effect on plant growth occurred at exogenous EE-GRSP at ½ strength. Similarly, the GRSP application also differently affected total chlorophyll content, root morphology (total length, surface area, and volume), and root N, P, and K content: positive effect by EE-GRSP and negative effect by DE-GRSP. Exogenous EE-GRSP accumulated more indoleacetic acid (IAA) in roots, which was associated with the upregulated expression of root auxin synthetic enzyme genes (PtTAA1, PtYUC3, and PtYUC4) and auxin influx transporter protein genes (PtLAX1, PtLAX2, and PtLAX3). On the other hand, exogenous DE-GRSP inhibited root IAA and indolebutyric acid (IBA) content, associated with the downregulated expression of root PtTAA1, PtLAX1, and PtLAX3. Root IAA positively correlated with root PtTAA1, PtYUC3, PtYUC4, PtLAX1, and PtLAX3 expression. Purified EE-GRSP and DE-GRSP showed similar element composition but varied in part element (C, O, P, Ca, Cu, Mn, Zn, Fe, and Mo) concentration. It concluded that exogenous GRSP triggered differential effects on growth response, and the effect was associated with the element content of pure GRSP and the change in auxins and root morphology. EE-GRSP displays a promise as a plant growth biostimulant in citriculture.

Effects of phosphorus, indolebutyric acid and naphthylphthalamic acid on the lateral root growth of Poncirus trifoliata

To explore the influence of phosphorus (P), indolebutyric acid (IBA, Auxin) and Naphthylphthalamic acid (NPA, Auxin transport inhibitor) on plant lateral root (LR) formation, Poncirus trifoliata seedlings at two P levels, low P (LP) and control treatment (CK), which was applied with IBA and NPA, and the regulative effects of P level, IBA and NPA on LR formation of trifoliate orange were investigated. The results showed that LP level significantly reduced the plant biomass, LR number and length. NPA significantly decreased the plant biomass, LR number and length, while IBA did not significantly influence these parameters. These data suggested that auxin signaling pathway could be involved in the regulation of P level on LR formation, and the auxin transportation should be the key factor in LR formation of trifoliate orange.

The Change in Fatty Acids and Sugars Reveals the Association between Trifoliate Orange and Endophytic Fungi

Endophytes have the ability to improve plant nutrition alongside their agronomic performance, among which arbuscular mycorrhizal fungi provide the most benefits to their host. Previously, we reported for the first time that an arbuscular mycorrhizal-like fungus Piriformospora indica had the ability to colonize roots of trifoliate orange (Poncirus trifoliata) and conferred positive effects on nutrient acquisition. Present study showed the changes in fatty acids and sugars to unravel the physiological and symbiotic association of trifoliate orange with P. indica and an arbuscular mycorrhizal fungus, Funneliformis mosseae singly or in combination. All the endophytic fungi collectively increased fructose, glucose, and sucrose content in leaves and roots, along with a relatively higher increase with P. indica inoculation than with F. mosseae alone or dual inoculation. Treatment with P. indica increased the concentration of part unsaturated fatty acids such as C18:3N6, C20:2, C20:3N6, C20:4N6, C20:3N3, C20:5N3, C22:1N9, and C24:1. Additionally, P. indica induced the increase in the concentration of part saturated fatty acids such as C6:0, C8:0, C13:0, C14:0, and C24:0. F. mosseae hardly changed the content of fatty acids, except for increase in C14:0 and C20:5N3. Double inoculation only reduced the C21:0, C10:0, C12:0, C18:3N3, and C18:1 content and increased the C20:5N3 content. These endophytic fungi up-regulated the root PtFAD2, PtFAD6, PtΔ9, and PtΔ15 gene expression level, coupled with a higher expression of PtFAD2 and PtΔ9 by P. indica than by F. mosseae. It was concluded that P. indica exhibited a stronger response, for sugars and fatty acids, than F. mosseae on trifoliate orange. Such results also reveal the Pi (an in vitro culturable fungus) as a bio-stimulator applying to citriculture.

PtrMYB3, a R2R3-MYB Transcription Factor from Poncirus trifoliata, Negatively Regulates Salt Tolerance and Hydrogen Peroxide Scavenging

MYB transcription factors are widely present in plants and play significant roles in abiotic stresses. However, most MYB genes have not been identified in plants and their functions in abiotic stresses are still unknown. In this study, one MYB gene, designated as PtrMYB3, was cloned from trifoliate orange (Poncirus trifoliata (L.) Raf.), and its function in salt tolerance was investigated. PtrMYB3 contains a conserved R2R3-MYB domain, which is the typical property of R2R3-MYB subfamily proteins. Expression profiling under abiotic stresses indicated that PtrMYB3 could be induced by salt, dehydration and cold stresses. PtrMYB3 was found to be localized to the nucleus and possessed transactivation activity. Overexpression of PtrMYB3 by genetic transformation in tobacco impaired its salt tolerance, whereas silencing of PtrMYB3 by VIGS (virus-induced gene silencing) in trifoliate orange conferred significantly enhanced salt tolerance, indicating that PtrMYB3 negatively regulates salt tolerance. Furthermore, a peroxidase gene (PtrPOD) was found to be greatly upregulated in PtrMYB3-silenced trifoliate orange, and a dual LUC (luciferase) assay confirmed that PtrMYB3 could suppress the expression of PtrPOD. The hydrogen peroxide (H2O2) accumulation in PtrMYB3 transgenic tobacco plants after salt stress was higher than the wild type (WT), further confirming that overexpression of PtrMYB3 inhibited PtrPOD-mediated H2O2 scavenging. Taken together, these results demonstrate that PtrMYB3 negatively regulates salt tolerance, at least in part, due to the excess accumulation of H2O2.