Chemical and microbiological changes in the soil mediated by different vegetative coverings in a Natal orange orchard

Proper soil cover management for citrus cultivation can contribute to increased productivity and improved soil quality. This study examined five different vegetative coverings [Urochloa brizantha; U. decumbens, U. ruziziensis, spontaneous vegetation, and herbicide application (glyphosate) in the total area] in the inter rows of a Natal orange orchard [Citrus sinensis (L.) Osbeck] grafted on the Swingle citrumelo (C. paradisi × Poncirus trifoliata). Their effects on the microbiological and chemical attributes of the soil and the vegetative development in the orchard were examined. Chemical (Ca2+, Mg2+, K+, P, pH, H+Al, CECpH7, base saturation, and OM) and microbiological (carbon and nitrogen of microbial biomass, basal respiration, and metabolic quotient) soil attributes in the rows and inter-rows were evaluated for the orchard in 2018 and 2019. There was a significant difference for most variables in the 2 years studied, emphasizing 2019 for microbiological parameters and OM, with the latter being 14.8% lower in the treatment with glyphosate in the total area compared to the treatment with spontaneous vegetation. The results showed the benefits of vegetation cover with brachiaria in inter-rows of the Natal sweet orange orchard in the chemical and microbiological attributes of the soil, especially in carbon and nitrogen of the microbial biomass.

First Report of citrus virus A in Texas associated with oak leaf patterns in Citrus sinensis

In 2018, Navarro et al. (2018a, b) identified two new negative sense coguviruses in citrus, citrus concave gum-associated virus (CCGaV) and citrus virus A (CiVA). Since then, the members of the genus Coguvirus have been also detected in other plant species (Xin et al. 2017; Wright et al. 2018; Svanella-Duma et al. 2019). In 2016, leaf flecking with oak leaf patterns were observed in five plants among embryo-rescued navel orange (NO) (Citrus sinensis (L.) Osbeck) trees grafted on C22 (C. sunki x Poncirus trifoliata) rootstock, maintained in a shade house. Madam Vinous (MV) sweet orange trees graft-inoculated with blind buds from the symptomatic NO plants developed the same symptoms in the new growth. These symptoms were similar to those on the citrus concave gum (CG) source tree of the California isolate CG301, one of the standard citrus disease isolates used as a positive control for biological indexing (Roistacher et al. 2000). None of the trees with oak leaf symptoms tested positive with reverse transcription (RT)-PCR for a panel of viruses and viroids commonly infecting citrus (Table S1) . In this study, CG301 leaf RNA-Seq data was used as a platform to identify any viral agent(s) associated with the oak leaf symptoms observed in the symptomatic NO trees (Fig. S1). Of ~162.8 million paired-end CG301 RNA-Seq reads (150 bp), de novo assembly of ~9.6 million reads, not mapped to C. sinensis genome (v.1.1), yielded 5,375 contigs. BLASTn using NCBI virus database (txid 10239) identified two contigs, #49 (6,715 nt) and #20 (2,764 nt), which exhibited ~96% sequence identity, respectively, to RNA1 and 2 of CiVA isolate W4 (MG764565; MG764566) and 71-73% identity to that of CCGaV isolate CGW2 (KX960112; KX960111). 5'-Nuclease assay developed based on contig #20 detected coguviral sequences in the five symptomatic NO and graft-inoculated MV trees as well as in CG301 but not in 44 asymptomatic field trees located near the shade house where the symptomatic NO trees had been kept. A full genomic sequence of the coguviruses present in CG301 and a symptomatic NO tree was reconstructed by RT-PCR. Both CG301 and NO isolate have a 6689 nt long negative sense RNA1 (MT922052; MK689372) encoding RNA-dependent RNA polymerase (RdRp) and a 2739 nt long ambisense RNA2 (MT922053; MK689373) encoding movement protein (MP) and nucleocapsid protein (NP). The isolate CG301 and NO share ~96% nucleotide sequence identity. The genome of both CG301 and NO isolate share 95.4-97.8 % sequence identity to that of CiVA isolate W4 and 70-72.9 % sequence identity to CCGaV isolate CGW2. BLASTp showed that RdRp of CG301 and NO isolate have 96.3-97.7 % sequence identity to CiVA W4 RdRp and ~77 % to CCGaV CGW2 RdRp. These data indicated the presence of CiVA in the symptomatic NO trees and in the concave gum source tree CG301. Recent reports of CiVA in South Africa and Greece indicated a potential wider distribution of CiVA in various citrus growing regions that may be associated with two graft-transmissible citrus diseases, citrus concave gum and impietratura disease (Roistacher et al. 2000; Velázquez et al. 2019; Beris et al. 2021; Bester et al. 2021). Although the source of CiVA in the symptomatic NO trees and the degree of CiVA prevalence in Texas had not been determined yet, a possible involvement of vector(s) or other means of spread (e.g. seed transmission) cannot be ruled out (Timmer et al. 2017). The current study demonstrated the need for further studies to determine the level of threat of coguviruses for citrus production in Texas.

Field Inoculation of Arbuscular Mycorrhizal Fungi Improves Fruit Quality and Root Physiological Activity of Citrus

Soil arbuscular mycorrhizal (AM) fungi form a mutualistic symbiosis with plant roots and produce many benefits on host plants under potted conditions, while field inoculation of AM fungi on citrus (a woody plant) has been rarely reported. The present study aimed to analyze the changes in mycorrhizal growth, root vitality, and fruit quality of Citrus reticulata Blanco var. Ponkan mandarin cv. Jinshuigan grafted on Poncirus trifoliata L. after inoculation with a mix of AM fungi (Diversispora versiformis, Funneliformis mosseae, and Rhizophagus intraradices) and single F. mosseae. After the second year of AM fungal inoculations, root mycorrhizal colonization (%), root vitality, hyphal length in soil, and easily extractable glomalin-related soil protein content were significantly increased, while difficult-to-extract glomalin-related soil protein content was decreased. Two mycorrhizal fungal inoculation treatments collectively improved fruit quality parameters such as polar diameter, equatorial diameter, the weight of single fruits, fruit peel, and sarcocarp, coloration value, and soluble solids content. Our study, therefore, suggested that field inoculation with AM fungi improved root physiological activities in terms of mycorrhizal growth and root vitality and thus improved fruit quality. The effect of mixed-AM treatment was more significant than that of F. mosseae alone.

Short-Term Waterlogging in Citrus Rootstocks

Changes in climate are provoking flooding events that cause waterlogging in the fields. Citrus are mainly cultivated in areas with a high susceptibility to climate change. Therefore, it is vital to explore their responses to these events to anticipate future challenges by means of genetic improvement of the commercial rootstocks. In this experiment, three popular commercial rootstocks, namely ‘Cleopatra’ (C. reshni Hort. Ex Tanaka), C. macrophylla, and ‘Forner Alcaide no. 5′ (Citrus reshni Hort. Ex Tanaka × Poncirus trifoliata), were evaluated after being submitted to short-term waterlogging and a period of recovery of 7 days in each case. Photosynthesis rate and stomatal conductance decreased in ‘Cleopatra’, while in the other two genotypes they were maintained (C. macrophylla) or restored after recovery (‘Forner Alcaide no. 5′). Relative water content and chlorophylls also decreased in ‘Cleopatra’. This indicates a deeper effect of flooding in ‘Cleopatra’, which suffered changes during flooding that were also sustained during the recovery phase. This did not occur in the other two rootstocks, since they showed signs of recovery for those parameters that decreased during waterlogging.

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.

Steam Explosion (STEX) of Citrus × Poncirus Hybrids with Exceptional Tolerance to Candidatus Liberibacter Asiaticus (CLas) as Useful Sources of Volatiles and Other Commercial Products

Florida citrus production has declined 75% due to Huanglongbing (HLB), a disease caused by the pathogenic bacterium Candidatus Liberibacter asiaticus (CLas). Methods to combat CLas are costly and only partially effective. The cross-compatible species Poncirus trifoliata and some of its hybrids are known to be highly tolerant to CLas, and thus can potentially serve as an alternative feedstock for many citrus products. To further investigate the commercial potential of citrus hybrids, three citrus hybrids, US-802, US-897, and US-942, were studied for their potential as feedstocks for citrus co-products using steam explosion (STEX) followed by water extraction. Up to 93% of sugars were recovered. US-897 and US-942 have similar volatile profiles to that of the commercial citrus fruit types and as much as 85% of these volatiles could be recovered. Approximately 80% of the pectic hydrocolloids present in all three hybrids could be obtained in water washes of STEX material. Of the phenolics identified, the flavanone glycosides, i.e., naringin, neohesperidin, and poncirin were the most abundant quantitatively in these hybrids. The ability to extract a large percentage of these compounds, along with their inherent values, make US-802, US-897, and US-942 potentially viable feedstock sources for citrus co-products in the current HLB-blighted environment.

Genome-wide identification and expression profiling of invertase gene family for abiotic stresses tolerance in Poncirus trifoliata

Background Sucrose (Suc) hydrolysis is directly associated with plants tolerance to multiple abiotic stresses. Invertase (INV) enzymes irreversibly catalyze Suc degradation to produce glucose (Glc) and fructose (Frc). However, genome-wide identification and function of individual members of the INV gene family in Poncirus trifoliata or its Citrus relatives in response to abiotic stresses are not fully understood. Results In this report, fourteen non-redundant PtrINV family members were identified in P. trifoliata including seven alkaline/neutral INV genes (PtrA/NINV1–7), two vacuolar INV genes (PtrVINV1–2), and five cell wall INV isoforms (PtrCWINV1–5). A comprehensive analysis based on the biochemical characteristics, the chromosomal location, the exon–intron structures and the evolutionary relationships demonstrated the conservation and the divergence of PtrINVs. In addition, expression analysis of INV genes during several abiotic stresses in various tissues indicated the central role of A/NINV7 among INV family members in response to abiotic stresses. Furthermore, our data demonstrated that high accumulation of Suc, Glc, Frc and total sugar contents were directly correlated with the elevated activities of soluble INV enzymes in the cold-tolerant P. trifoliata, C. ichangensis and C. sinensis, demonstrating the potential role of soluble INV enzymes for the cold tolerance of Citrus. Conclusions This work offered a framework for understanding the physiological role of INV genes and laid a foundation for future functional studies of these genes in response to abiotic stresses.

Gene Expression under Short-Term Low Temperatures: Preliminary Screening Method to Obtain Tolerant Citrus Rootstocks

Climate change and global warming are leading to a change in weather patterns toward hot and cold waves. Citrus fruits are a tropical or subtropical crop whose growth is altered by changes in weather patterns. Thus, in the present work, two experiments are evaluated to obtain a screening method to select citrus rootstocks that help us to select new low-temperature-tolerant plant materials. One cold experiment was carried out with the Poncirus trifoliata and Citrus macrophylla rootstocks at 4 °C for 4, 8, 24 and 56 h. A second experiment was performed at 4 °C for 5 days with subsequent acclimatization lasting 0, 5, 10 and 24 h. The expression of the cold response genes CAMTA1, CAMTA3, CAMTA5, CBF1, ICE1 and COR413 IM1 was quantified. The results showed that the best rootstock selection strategy was the second experiment, as a higher expression of the genes CAMTA3, CAMTA5, CBF1 and COR413 IM1 was seen in the tolerant genotype P. trifoliata. We quantified the gene expression of proline biosynthesis P5CS1, dOAT and the proline transporters PROT1 and PROT2; the concentration of the amino acid proline in leaves was also quantified. These results once again showed that the best experiment to differentiate between tolerant and sensitive rootstocks was the second experiment with acclimation time.

Development of a Simultaneous Analysis Method for Quality Control of a Traditional Herbal Formula, Daeshiho-Tang, Using 10 Marker Components

Daeshiho-tang (DSHT) is a traditional herbal formula consisting of six herbal medicines: Bupleurum falcatum L., Scutellaria baicalensis Georgi, Paonia lactiflora Pall., Pheum palmatum L., Poncirus trifoliata (L.) Raf., and Pinellia ternate (Thunb.) Makino. In this study, we developed a simultaneous analysis method based on high-performance liquid chromatography for the quality control of DSHT. Chromatographic separation of 10 marker components (gallic acid, albiflorin, paeoniflorin, naringin, benzoic acid, baicalin, poncirin, wogonoside, baicalein, and wogonin) was achieved using a water–acetonitrile system as the mobile phase with a SunFire C18 reversed-phase column. The developed analytical method was validated with respect to linearity, limit of detection, limit of quantitation, recovery, and precision. Among the 10 markers of DSHT in the established assay, baicalin, the main compound of Scutellaria baicalensis Georgi, was present in the highest concentration (36.86–46.17 mg/g). The validated assay will be useful for the quality control of DSHT.

Screening of ‘King’ Mandarin Hybrids as Tolerant Citrus Rootstocks to Flooding Stress

This work compares the tolerance to long-term anoxia conditions (35 days) of five new citrus ‘King’ mandarin (Citrus nobilis L. Lour) × Poncirus trifoliata ((L.) Raf.) hybrids (named 0501XX) and Carrizo citrange (CC, Citrus sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.), the widely used citrus rootstock in Spain. Growth parameters, chlorophyll concentration, gas exchange and fluorescence parameters, water relations in leaves, abscisic acid (ABA) concentration, and PIP1 and PIP2 gene expressions were assessed. With a waterlogging treatment, the root system biomass of most hybrids went down, and the chlorophyll a and b concentrations substantially dropped. The net CO2 assimilation rates (An) and stomatal conductance (gs) lowered significantly due to flooding, and the transpiration rate (E) closely paralleled the changes in gs. The leaf water and osmotic potentials significantly increased in most 0501 hybrids. As a trend, flooding stress lowered the ABA concentration in roots from most hybrids, but increased in the leaves of CC, 05019 and 050110. Under the control treatment (Ct) conditions, most 0501 hybrids showed higher PIP1 and PIP2 expressions than the control rootstock CC, but were impaired due to the flooding conditions in 05019 and 050110. From this study, we conclude that 0501 genotypes develop some adaptive responses in plants against flooding stress such as (1) stomata closure to prevent water loss likely mediated by ABA levels, and (2) enhanced water and osmotic potentials and the downregulation of those genes regulating aquaporin channels to maintain water relations in plants. Although these traits seemed especially relevant in hybrids 050110 and 050125, further experiments must be done to determine their behavior under field conditions, particularly their influence on commercial varieties and their suitability as flooding-tolerant hybrids for replacing CC, one of the main genotypes that is widely used as a citrus rootstock in Spain, under these conditions.