Distribution and genetic diversity of Coguvirus eburi in South African citrus and the development of a real-time RT-PCR assay for citrus-infecting coguviruses

Citrus virus A (CiVA), a novel negative-sense single-stranded RNA virus assigned to the species Coguvirus eburi in the genus Coguvirus, was detected in South Africa with the use of high-throughput sequencing (HTS) after its initial discovery in Italy. CiVA is closely related to citrus concave gum-associated virus (CCGaV), recently assigned to the species Citrus coguvirus. Disease association with CiVA is however incomplete. CiVA was detected in grapefruit (Citrus paradisi Macf.), sweet orange (C. sinensis (L.) Osb.) and clementine (C. reticulata Blanco) in South Africa and a survey to determine the distribution, symptom association and genetic diversity was conducted in three provinces and seven citrus production regions. The virus was detected in ‘Delta’ Valencia trees in six citrus production regions and a fruit rind symptom was often observed on CiVA-positive trees. Additionally, grapefruit showing symptoms of citrus impietratura disease were positive for CiVA. This virus was primarily detected in older orchards that were established prior to the application of shoot tip grafting for virus elimination in the South African Citrus Improvement Scheme. The three viral encoded genes of CiVA isolates from each cultivar and region were sequenced to investigate sequence diversity. Genetic differences were detected between the ‘Delta’ Valencia, grapefruit and clementine samples, with greater sequence variation observed with the nucleocapsid protein (NP) compared to the RNA-dependent RNA polymerase (RdRp) and the movement protein (MP). A real-time detection assay, targeting the RdRp, was developed to simultaneously detect citrus infecting coguviruses, CiVA and CCGaV, using a dual priming reverse primer to improve PCR specificity.

Correlation of Soil Characteristics and Citrus Leaf Nutrients Contents in Current Scenario of Layyah District

Soil with low fertility is a big problem for achieving citrus productivity. In this regard, the management of macro and micronutrients is essential. Macro and micronutrient deficiency decreased the yield and the quality of citrus fruit. It is the need of the hour to classify the soil fertility status under changing climatic scenarios. The current soil fertility survey was conducted to examine the macro and micronutrient status in the citrus production area. In soil, three depths (0–15, 15–30, and 30–45 cm) were taken for sampling. For leaves, 4–6-months-old non-bearing twigs were sampled from 20 trees per orchard at breast height. Results showed that soil pH (7.1–8.4) was slightly alkaline, electrical conductivity (EC) was non-saline (<4 dSm−1), soil organic matter (SOM) was deficient (<0.86%), and calcium carbonate (CaCO3) was slight calcareous (<8%), at 0–15, 15–30, and 30–45 cm depths. The majority of soil samples were low in nitrogen (N) contents at all depths, i.e., (<0.043) 0–15 (85%), 15–30 (97%), and 30–45 (100%) cm depths. Phosphorus (P) was medium (7–15 mg kg−1) at 0–15 cm (60%) but low (<7 mg kg−1) at 15–30 (63%) and 30–45 cm (82%) depths. Potassium (K) was medium (80–180 mg kg−1) at 0–15 (69%), 15–30 (69%), and 30–45 cm (10%) depths. Boron (B) and manganese (Mn) were medium, and Cu was high in 0.15 cm, but all were low at 15–30 and 30–45 cm depths. Iron (Fe) and zinc (Zn) were low at depths of 0–15, 15–30, and 30–45 cm. Most citrus leaves were deficient in N (94%), Fe (76%), Zn (67%), and B (67%). In conclusion, soil fertilization is not sufficient for optimum citrus yield because of alkaline pH and slight calcareous soil conditions in this region. Foliar application of nutrients is suggested instead of only soil fertilization, for better nutrient management in citrus orchards.

Different Sweet Orange–Rootstock Combinations Infected by Candidatus Liberibacter asiaticus under Greenhouse Conditions: Effects on the Scion

The devastating citrus disease huanglongbing (HLB) associated with the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas) has caused a more than 70% reduction in citrus production since its discovery in Florida in 2005. Most citrus scion cultivars are sensitive to HLB, whereas some cultivars used as rootstocks are tolerant. Using such tolerant rootstocks can help trees to cope better with the disease’s impact. Evaluating rootstock effects on a grafted scion in the field takes many years, but shorter-term evaluation is imperative to aid in rootstock selection for an HLB-endemic production environment. In this study, we investigated grafted healthy and CLas-infected citrus trees under controlled greenhouse conditions. The objectives were to identify traits suitable for assessing grafted tree tolerance in advance of longer-term field studies and aiding in the selection of superior rootstock cultivars. We assessed 10 commercially important rootstocks grafted with ‘Valencia’ sweet orange scion and with known field performance. At 6, 9, 15, and 21 months after graft inoculation (mai), leaf CLas titers were determined and canopy health was evaluated. Plants were destructively sampled at 21 mai to assess plant biomasses and other physiological and horticultural variables. There was little influence of the rootstock cultivar on CLas titers. Surprisingly, few HLB foliar disease symptoms and no differences in soluble and nonsoluble carbohydrate concentrations were measured in infected compared with healthy plants, despite high CLas titers and significant reductions in plant biomasses. Most trees on rootstocks with trifoliate orange parentage were less damaged by HLB than other rootstocks, although results did not always agree with reported field performance. Among the different variables measured, leaf size appeared to be most predictive for grafted tree assessment of HLB sensitivity. The results of this study provide a better understanding of the strengths and weaknesses of assessing rootstock influence on grafted tree performance in a controlled greenhouse environment. Although such studies provide valuable information for cultivar tolerance to HLB, other rootstock traits will ultimately contribute to field survival and productivity in an HLB endemic production environment.

Understanding Citrus Greening Disease and Its Possible Management Strategies in Nepal

Huanglongbing (HLB), also known as citrus greening, is a devastating disease of citrus that has decimated several citrus orchards throughout the world. The disease is associated with three species of unculturable and phloem-limited bacteriae, Candidatus Liberibacter asiaticus, Candidatus Liberibacter africanus and Candidatus Liberibacter americanus. The most common species of bacteria found in Nepal is Candidatus Liberibacter asiaticus which is transmitted by an insect vector, Asian citrus psyllid (Diaphorina citri). This disease has been detected in several economically important citrus production areas of Nepal, which resulted in heavy yield loss. No cure for the disease has been discovered yet and it is essential to practice proper management strategies to maintain citrus health and sustain citrus production under HLB pressure. Several disease management approaches such as pathogen-free nursery establishment, use of disease tolerant rootstock cultivars, proper irrigation and nutrient supply, removal of HLB affected trees, and control of psyllid with frequent insecticide application are widely practiced throughout the world. This review article highlights the characteristics of the citrus greening disease and its insect vector and gives insights into their management techniques. Several technologically advanced options available to minimize the HLB infection might not be feasible currently in Nepal due to economic and topographic constraints. This article also aims to bring into focus the cost-effective methods that growers in Nepal can practice to mitigate the impact of HLB disease in their citrus orchards. Int. J. Appl. Sci. Biotechnol. Vol 9(4): 227-238.

Role of Plant Volatile to Diaphorina citri on Feeding and Oviposition Behaviour

Citrus production in Indonesia and in the world is suffered from disease of citrus vein phloem degeneration (CVPD. It was vectored by psylids (Diaphorina citri). The psylids used citrus plant volatiles as cues for finding their host plants for feeding and laying eggs. Extract of guava leaves was a prospective control material for declaining population of psyllid. Investigation by research was conducted to determine the effect of CVPD symptomatic citrus plants in attracting psylids gravid female for staying and laying eggs, and also to determine the repellency ability of guava shoots to adult psyllids. Citrus buds of healthy plants and CVPD symptomatic plants were exposed to ten gravid female and the number of psylids stay and the number of eggs per bud was recorded. Y-tube olfactometer was used to determine repellent effect of upper shoot, middle shoot, and bottom shoot of guava leave to ten adult psyllids. The result shows that CVPD symptomatic plants was more attractive for laying eggs, eventhough the number of eggs was higher on healthy plants. Repellence effect to psyllids adult was identified in guava leave extracts. The increase of leaf age would decrease the effect. It was suggested that repellent properties was highest youngest leaf or upper shoots of guava.

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.

Techniques for Reducing the Abundance of Spring–Summer Flush Shoots in Southern Spanish Orange Orchards

The Mediterranean Basin is the second highest citrus growing region in the world behind China. Citrus trees are known to produce several flush shoots per year, particularly during the spring–summer season. Farmers endeavor to reduce the growth of summer shoots by means of hand pruning, especially those located at the top of the tree, as most of these shoots become vigorous, nutrient consuming, non-productive, and attractive to several pests. Furthermore, hand pruning substantially increases the costs of citrus orchards production. This research was therefore intended to study new different treatments to control spring–summer flush shoots and thus reduce growers’ investments in citrus production. Six different treatments were applied in two experimental and high density orange orchards over two consecutive years: (1) control; (2) topping (mechanical pruning); (3) dichlorprop-p; (4) triclopyr; (5) topping + dichlorprop-p; and (6) topping + triclopyr. The treatment of dichlorprop-p alone reduced the number of summer young shoots in both years. Moreover, these applications did not negatively affect yield or fruit quality. These mechanical methodologies help citrus growers manage the density of flush shoots and reduce hand labor costs in citrus orchards.

PHYLOGENY AND MORPHOLOGY OF NEODIDYMELLIOPSIS IRANENSIS SP. NOV., CAUSING DEAD BRANCHES OF CITRUS IN IRAN

Citrus plants, which are mostly cultivated in the southern tropical area of the country, are one of the important economic crops in Iran. Branch canker and dieback of citrus is an ongoing problem for citrus growers located in these areas and has imposed irreparable damage to the citrus production in this region in recent years. Disease symptoms consisted of the blight of vigorously growing shoots and dieback of the branches and rootstock trunks. This study aimed to characterize the citrus dieback pathogen morphologically and phylogenetically, and the species Neodidymelliopsis iranensis Soleimani & Goudarzi, sp. nov., is described and illustrated here. Isolates were derived from collected citrus samples with dieback symptoms. After preparing pure cultures from single spores on oatmeal agar and malt agar, the morphological features of the species were described and their pathogenicity was confirmed on lime (Citrus aurantifolia). Morphologically N. iranensis is easily separated from the other species of Neodidymelliopsis by the size of pycnidia, conidia septation, and NaOH test results. The morphological differences between our isolates and the other known species of Neodidymelliopsis were strongly supported by a multi-locus phylogenetic analysis based on the ITS region, and LSU, RPB2, and TUB2 genes. In the reconstructed phylogenetic tree, N. iranensis formed a well-supported clade with other Neodiddymelliopsis species in the Didymellaceae family, but was separated from all other Neodiddymelliopsis species. The distinct phylogenetic position is supported by differences in morphological features. Consequently, the specificity of the morphological and phylogenetical features of the collected isolates has convinced us to describe Neodidymelliopsis iranensis as a new species.

Predation of Florida red scale Chrysomphalus aonidum (L.) by the metallic blue ladybeetle Curinus coeruleus (Mulsant)

Florida red scale, Chrysomphalus aonidum (L.) is a species of armored scales, which attacks citrus crops. Biological control, particularly ladybeetles, are critical for its management in citrus production systems. We evaluated predation of C. aonidum by adult and larvae (3rd–4th instar) of the metallic blue ladybeetle, Curinus coeruleus. C. coeruleus were tested against C. aonidum in three separate treatments on Valencia orange leaves in Petri-dish arenas (1) individuals with armor intact, (2) individuals with armor removed, and (3) mix of individuals with armor intact or removed, to determine if armor inflicts distraction to predator consumption of scale and impact. Within 24 h of exposure to C. aonidum, adult beetle consumption rate averaged 64–68% in the two treatments containing all or half of the individuals with armor, compared to 100% in the treatment with armor removed. The consumption rate in the former two treatments increased to 83–89% within 72 h. Larval consumption of C. aonidum with armor intact was 25% at 24 h and 41% at 72 h, compared to 92–100% in the treatment with armor removed. The adults resulting from the larvae developed on C. aonidum with the armor intact or between the diets of armor intact or removed consumed more scales without armor than with armor. Significant consumption of C. aonidum by adults and larvae of C. coeruleus indicates that it is an efficient predator of this pest species. These novel findings suggest that this predator could be useful for suppressing C. aonidum populations in citrus production systems, particularly in habitats where both species are established such as Florida.