Non-target effects of ten essential oils on the egg-parasitoid Trichogramma evanescens

Essential oils (EOs) are increasingly used as biopesticides due to their insecticidal potential. This study addresses their non-target effects on a biological control agent: the egg parasitoid Trichogramma evanescens. In particular, we tested whether EOs affected parasitoid fitness either directly, by decreasing pre-imaginal survival, or indirectly, by disrupting parasitoids' orientation abilities. The effect of Anise, Fennel, Sweet orange, Basil, Coriander, Oregano, Peppermint, Mugwort, Rosemary and Thyme EOs were studied on five strains of T. evanescens. Specific experimental setups were developed, and data obtained from image analysis were interpreted with phenomenological models fitted with Bayesian inference. Results highlight the fumigant toxicity of EOs on parasitoid development. Anise, Fennel, Basil, Coriander, Oregano, Peppermint and Thyme EOs are particularly toxic and drastically reduce the emergence rate of T. evanescens. Most EOs also affect parasitoid behavior: (i) Basil, Coriander, Oregano, Peppermint, Mugwort and Thyme EOs are highly repellent for naive female parasitoids; (ii) Anise and Fennel EOs can have repellent to attractive effects depending on strains; and (iii) Sweet orange, Oregano and Rosemary EOs have no detectable impact on orientation behavior. This study shows that EOs fumigation have non-target effects on egg parasitoids. This highlights the need to cautiously precise the deployment framework of biopesticides in an agroecological perspective.

Genetic diversity and population structure of sweet orange [Citrus sinensis (L.) Osbeck] germplasm of India revealed by SSR and InDel markers

Sweet orange (Citrus sinensis (L.) Osbeck) is an important commercial citrus fruit crop, cultivated in India and across the world. In India most of the cultivated sweet orange species were introduced varieties. In this study, we used two molecular markers, SSR and InDels, to understand the genetic diversity and population structure of seventy-two sweet orange genotypes. Genetic parameters consisted of a total number of alleles, a number of polymorphic alleles (effective alleles); genetic diversity (G.D.), expected heterozygosity (He), and the polymorphic information content (PIC) were calculated based on molecular data. Two dendrograms were constructed based on the InDels and SSR. In both the cases, they formed three major clusters showing various degrees of variations with respect to members of the clusters. Population structure analysis revealed the presence of two distinct subpopulations. Therefore, in order to address various challenges and develop sweet orange varieties with desirable traits, there is a need to broaden the genetic base of sweet orange through the intensive collection in the northeastern region. These results of intraspecific genetic variability of the collections will dictate the path for the sweet orange breeding and conservation programs in India.

Highly Efficient Generation of Canker-Resistant Sweet Orange Enabled by an Improved CRISPR/Cas9 System

Sweet orange (Citrus sinensis) is the most economically important species for the citrus industry. However, it is susceptible to many diseases including citrus bacterial canker caused by Xanthomonas citri subsp. citri (Xcc) that triggers devastating effects on citrus production. Conventional breeding has not met the challenge to improve disease resistance of sweet orange due to the long juvenility and other limitations. CRISPR-mediated genome editing has shown promising potentials for genetic improvements of plants. Generation of biallelic/homozygous mutants remains difficult for sweet orange due to low transformation rate, existence of heterozygous alleles for target genes, and low biallelic editing efficacy using the CRISPR technology. Here, we report improvements in the CRISPR/Cas9 system for citrus gene editing. Based on the improvements we made previously [dicot codon optimized Cas9, tRNA for multiplexing, a modified sgRNA scaffold with high efficiency, citrus U6 (CsU6) to drive sgRNA expression], we further improved our CRISPR/Cas9 system by choosing superior promoters [Cestrum yellow leaf curling virus (CmYLCV) or Citrus sinensis ubiquitin (CsUbi) promoter] to drive Cas9 and optimizing culture temperature. This system was able to generate a biallelic mutation rate of up to 89% for Carrizo citrange and 79% for Hamlin sweet orange. Consequently, this system was used to generate canker-resistant Hamlin sweet orange by mutating the effector binding element (EBE) of canker susceptibility gene CsLOB1, which is required for causing canker symptoms by Xcc. Six biallelic Hamlin sweet orange mutant lines in the EBE were generated. The biallelic mutants are resistant to Xcc. Biallelic mutation of the EBE region abolishes the induction of CsLOB1 by Xcc. This study represents a significant improvement in sweet orange gene editing efficacy and generating disease-resistant varieties via CRISPR-mediated genome editing. This improvement in citrus genome editing makes genetic studies and manipulations of sweet orange more feasible.

Influence of probiotics on physico-chemical and organoleptic characteristics of sweet orange juice

Aim: The study was undertaken to evaluate the survival probiotic organisms and its influence on the physical, chemical, nutritional and sensory characteristics of sweet orange juice. Methodology: Two samples of probiotic juice were prepared with 10 percent innoculum containing LAB strains (Lactobacillus bulgaricus and Lactobacillus plantarum). Sample A (without encapsulated strains) and Sample-B (with encapsulated strains) were prepared and incubated for 10hrs at 35oC. After incubation, the physico-chemical analysis of both the samples were analyzed for TSS, pH, acidity, total sugars, reducing sugars and ascorbic acid content. Results: The results of TSS, pH, acidity, total sugars, reducing sugars and ascorbic acid content for sample –A and Sample –B were 11.4˚Brix, 3.51, 0.82 percent, 6.1 percent, 1.5 percent, 4.6 percent, 40mgml-1 and 11.6˚ Brix, 3.68, 0.77 percent, 6.4 percent, 1.7 percent, 4.9 percent, 40 mg ml-1, respectively. Sensory evaluation revealed that overall acceptance of probiotic juice containing encapsulated strains and free strains in the first week was 8.3 and 7.8, respectively. Even after 4 weeks of storage, the overall acceptance for juice with encapsulated strains was better than free strains with a score of 7.5 and 7.0 at the end of storage period. Interpretation: The sweet orange juice with encapsulated strains has high viable cell count (109cfu ml-1) even after 4 weeks of storage resulted in stable therapeutic probiotic sweet orange juice. It is further, suitable for commercial production of probiotic sweet orange juice with probiotic cultures.

Graft Compatibility Classification within Aurantioideae Based on Biometric Traits and the Anatomy of Graft Union

Citrus relatives are a relevant source of valuable traits for use in citrus breeding, including resistance to diseases such as Huanglongbing (HLB). Resistant rootstocks may impact tree responses to HLB. This requires graft compatibility, which has been poorly investigated within the Aurantioideae. In this study, the biometric characteristics and the anatomy of the graft union of 86 scion/rootstock combinations were assessed. This comprised 18 genotypes/species and 8 genera from Citrinae, Balsamocitrinae, and Clauseninae subtribes sensu Swingle and Reece. Most graft combinations were found to be noncompatible. Phylogenetic proximity did not ensure successful grafting as, for example, Orange jasmine autografts failed, whereas some intergeneric grafts were successful (>60% of graft-take). Plant scion height was directly related to graft-take, but the correlation between the scion and rootstock stem diameters was not a reliable indicator of graft compatibility. Rangpur/Tabog, Tabog/Rangpur, Wampee/Rangpur, Wampee/Pomeroy, Wampee/Swingle, Pomeroy/Wampee, and Swingle/Wampee were the most compatible intergeneric graft combinations. Graft-take success for this was at similar levels to those of sweet orange grafted on common citrus rootstocks. The position as a scion or rootstock in the combination affected the performance and was specific to the genotypes tested. The lack of differentiation between xylem-derived calli and the accumulation of phenolic compounds at the graft union were clear anatomical and biochemical markers, respectively, of incompatibility for most Aurantioideae combinations. In the field, within a set of the ten most promising combinations, Hamlin/Rangpur (control) was the only one that became infected by ‘Candidatus Liberibacter asiaticus’. This was first observed 12 months after planting. Overall, the assessment of biometric traits and anatomy of the graft union allowed Aurantioideae genotypes to be divided into four clusters, with respect to their graft compatibility, as follows: fully compatible with high graft-take and plant growth; potentially compatible with high graft-take but lower plant growth; partially incompatible with lower graft-take and poor plant growth; and fully incompatible with a complete absence of graft-take.

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.