First Report of ‘CandidatusLiberibacter asiaticus’ Associated with Huanglongbing in Sweet Orange in Ethiopia

Huanglongbing (HLB) is a serious disease of citrus worldwide. Three different ‘Candidatus Liberibacter’ species are associated with HLB: ‘Ca. Liberibacter asiaticus’, ‘Ca. L. africanus’, and ‘Ca. L. americanus’ (1). ‘Ca. L. africanus’ and its vector, Trioza erytreae, are both heat sensitive, and when present, occur in citrus when temperatures remain below 30 to 32°C. In Africa, ‘Ca. L. africanus’ and T. erytreae have been reported in South Africa, Zimbabwe, Malawi, Burundi, Kenya, Somalia, Ethiopia, Cameroon, and Madagascar (1). Inspection of citrus trees in orchards and budwood sources in nurseries located in the warmer citrus-growing areas of Tigray and North Wollo in northern Ethiopia revealed nearly 100 trees with symptoms of leaf yellowing with a blotchy mottle pattern, dead branches, and decreased fruit quality and yield. Two symptomatic sweet orange budwood trees and three symptomatic orchard plants were sampled in April 2009, along with three healthy-looking sweet orange plants. DNA was extracted from 200 mg of desiccated leaf midribs using the CTAB method (4) and subjected to conventional PCR using the primer pairs A2/J5 (2) and OI2/23S1 (3) that amplify the ribosomal protein gene in the rplKAJL-rpoBC operon and the 16S/23S ribosomal intergenic regions, respectively, of ‘Ca. L. africanus’ and ‘Ca. L. asiaticus’. Positive PCR reactions were obtained for all five symptomatic samples with both primer pairs. PCR amplicons of 703 bp (A2/J5) and 892 bp (OI2/23S) recovered from two of these samples were purified, cloned, and sequenced. BLAST analysis revealed that the nucleotide sequences we obtained for the ribosomal protein (GenBank Accessions Nos. GQ890155 and GQ890156) shared 100% identity with each other and 99% identity with sequences of ‘Ca. L. asiaticus’ from Brazil (DQ471904), Indonesia (AB480161), China (DQ157277), and Florida (CP001677). Similarly, the 16S/23S ribosomal intergenic sequences (GU296538 and GU296539) shared 100% identity with each other and 99% identity with homologous ‘Ca. L. asiaticus’ sequences from Brazil (DQ471903), Indonesia (AB480102), China (DQ778016), and Florida (CP001677) and contained two tRNA genes as occurs in ‘Ca. L. asiaticus’ but not in ‘Ca. L. africanus’ (3). To our knowledge, this is the first report of ‘Ca. L. asiaticus’ in Africa. The presence of ‘Ca. L. asiaticus’ is a threat for warmer citrus-growing areas of Africa that are less favorable for ‘Ca. L. africanus’ and T. erytreae. In areas where ‘Ca. L. asiaticus’ was confirmed, symptomatic trees must be promptly eradicated and surveys to determine spread of the disease and its vectors are necessary

Non-lethal effects of N-acetylcysteine on xylella fastidiosa strain De Donno biofilm formation and detachment

This study investigated in-vitro the non-lethal eects of N-acetylcysteine (NAC) on Xylella fastidiosa subspecies pauca strain De Donno (Xf-DD) biofilm. This strain was isolated fromthe olive trees aected by the olive quick decline syndrome in southern Italy. Xf-DD was first exposed to non-lethal concentrations of NAC from 0.05 to 1000 M. Cell surface adhesion was dramatically reduced at 500 M NAC (47%), hence, this concentration was selected for investigating the eects of pre-, postand co-treatments on biofilm physiology and structural development, oxidative homeostasis, and biofilm detachment. Even though 500 MNAC reduced bacterial attachment to surfaces, compared to the control samples, it promoted Xf-DD biofilm formation by increasing: (i) biofilm biomass by up to 78% in the co-treatment, (ii) matrix polysaccharides production by up to 72% in the pre-treatment, and (iii) reactive oxygen species levels by 3.5-fold in the co-treatment. Xf-DD biofilm detachment without and with NAC was also investigated. The NAC treatment did not increase biofilm detachment, compared to the control samples. All these findings suggested that, at 500 M, NAC diversified the phenotypes in Xf-DD biofilm, promoting biofilm formation (hyper-biofilm-forming phenotype) and discouraging biofilm detachment (hyper-attachment phenotype), while increasing oxidative stress level in the biofilm

Embryo Culture, In Vitro Propagation, and Molecular Identification for Advanced Olive Breeding Programs

The high biodiversity of the olive tree is an important opportunity to develop sustainable plans to control Xylella fastidiosa (X) through breeding programs. Olive tree breeding activities have been limited due to various features of this species including the long time required for seed germination caused by the inhibition effect of the woody endocarp, the seed integument, and the endosperm. Starting from F1 seeds by cross-breeding, the embryo culture was compared with traditional seed germination, evaluating the effectiveness of in vitro multiplication of the plantlets for large-scale production. The isolated embryos were established on a new medium based on Rugini ‘84 macroelements, Murashige & Skoog ‘62 microelements, with Nitsch J. P. & Nitsch C. ‘69 vitamine and subcultured on Leva MSM modified. The results obtained confirmed that in vitro culture of olive embryos is a valid tool for increasing the percentage and speed of germination, helping to reduce the time of the olive breeding programs, offering the possibility to effectively propagate plantlets for further experiments

A non-targeted metabolomics study on Xylella fastidiosa infected olive plants grown under controlled conditions

In the last decade, the bacterial pathogen Xylella fastidiosa has devastated olive trees throughout Apulia region (Southern Italy) in the form of the disease called “Olive Quick Decline Syndrome” (OQDS). This study describes changes in the metabolic profile due to the infection by X. fastidiosa subsp. pauca ST53 in artificially inoculated young olive plants of the susceptible variety Cellina di Nardò. The test plants, grown in a thermo-conditioned greenhouse, were also co-inoculated with some xylem-inhabiting fungi known to largely occur in OQDS-affected trees, in order to partially reproduce field conditions in terms of biotic stress. The investigations were performed by combining NMR spectroscopy and MS spectrometry with a non-targeted approach for the analysis of leaf extracts. Statistical analysis revealed that Xylella-infected plants were characterized by higher amounts of malic acid, formic acid, mannitol, and sucrose than in Xylella-non-infected ones, whereas it revealed slightly lower amounts of oleuropein. Attention was paid to mannitol which may play a central role in sustaining the survival of the olive tree against bacterial infection. This study contributes to describe a set of metabolites playing a possible role as markers in the infections by X. fastidiosa in olive

Screening of olive germplasm for resistance to Xylella fastidiosa ST53: the state of the art

While different sources of natural resistance to X. fastidiosa have been described in grapevines and citrus, lack of solid information exists on possible sources of resistance/tolerance in the cultivars that characterize the wide olive germplasm. Preliminary field observations and laboratory analyses of a few cultivars, have shown that differential responses to X. fastidiosa infections exist. To confirm these preliminary findings, a large panel of olive cultivars is being specifically investigated. Currently, the screening procedure relies on field observations looking for symptomless subjects (trees of known cultivars/volunteer seedlings), mechanical inoculations, qualitative and quantitative diagnostic assays (ELISA & qPCR) and, in selected cases, comparative transcriptomic profiling. Field experiments include the planting of the target cultivars/selections in an infected area under high inoculum pressure. All the plots are located in the Apulia Region (Italy) in the demarcated infected area, surrounded by X. fastidiosa heavily affected olive groves. A first experimental plot was established in April 2015 with 10 different cultivars, which was extended in 2016 to 49 cultivars, and will be further enlarged in 2017 with the addition of 40 new accessions. Other plots, comprising newly planted or grafted cultivars (over 260 cvs) have been also established, bringing to over 300 the total number of accessions under evaluation. Cvs Leccino and FS-17®, both expressing interesting traits of resistance, have already been identified

Progress and achievements on the early detection of Xylella fastidiosa infection and symptom development with hyperspectral and thermal remote sensing imagery

Trabajo presentado en la 3rd European Conference on Xylella fastidiosa (Building knowledge, protecting plant health), celebrada online el 29 y 30 de abril de 2021.Remote sensing efforts made as part of European initiatives via POnTE, XF-ACTORS and the JRC, as well as through regional programs, have focused, among others, on the development of algorithms for the early detection of Xylella fastidiosa (Xf)-induced symptoms. Airborne campaigns carried out between 2016 and 2019 collected high-resolution hyperspectral and thermal images from infected areas in the Apulia region (Italy), in the province of Alicante and on the island of Mallorca (Spain). The remote sensing imagery collections were performed alongside field surveys and laboratory analyses to assess the presence of Xf, and the severity and incidence of disease in olive and almond trees. Radiative transfer models and machine learning algorithms were used to quantify spectral plant traits for each individual infected tree, assessing their importance as pre visual indicators of Xf-induced stress. These studies conducted across species have demonstrated that specific spectral plant traits successfully revealed Xf induced symptoms at early stages, i.e., before visual symptoms appear. The results show that spectral plant traits contribute differently to symptom detection across host species (olive vs. almond), and that abiotic-induced stress affects the performance of the algorithms used for detecting infected trees. Together, the different European initiatives studying the use of remote sensing to support the monitoring of landscapes for Xylella fastidiosa detection lead us to conclude that the early detection of Xf-induced symptoms is feasible when high-resolution hyperspectral imagery and physically-based plant trait retrievals are used, obtaining accuracies exceeding 92% (kappa>0.8). These results are essential to enable the implementation of effective control and management of plant diseases using airborne- droneand satellite-based remote sensing technologies. Moreover, these large-scale hyperspectral and thermal imaging methods greatly contribute to the future operational monitoring of infected areas at large scales, well beyond what is possible from field surveys and laboratory analyses alone