Detection of Xylella fastidiosa in almond orchards by synergic use of an epidemic spread model and remotely sensed plant traits.

The early detection of Xylella fastidiosa (Xf) infections is critical to the management of this dangerous plan pathogen across the world. Recent studies with remote sensing (RS) sensors at different scales have shown that Xf-infected olive trees have distinct spectral features in the visible and infrared regions (VNIR). However, further work is needed to integrate remote sensing in the management of plant disease epidemics. Here, we research how the spectral changes picked up by different sets of RS plant traits (i.e., pigments, structural or leaf protein content), can help capture the spatial dynamics of Xf spread. We coupled a spatial spread model with the probability of Xf-infection predicted by a RS-driven support vector machine (RS-SVM) model. Furthermore, we analyzed which RS plant traits contribute most to the output of the prediction models. For that, in almond orchards affected by Xf (n = 1426 trees), we conducted a field campaign simultaneously with an airborne campaign to collect high-resolution thermal images and hyperspectral images in the visible-near-infrared (VNIR, 400-850 nm) and short-wave infrared regions (SWIR, 950-1700 nm). The best performing RS-SVM model (OA = 75%; kappa = 0.50) included as predictors leaf protein content, nitrogen indices (NIs), fluorescence and a thermal indicator (Tc), alongside pigments and structural parameters. Leaf protein content together with NIs contributed 28% to the explanatory power of the model, followed by chlorophyll (22%), structural parameters (LAI and LIDFa), and chlorophyll indicators of photosynthetic efficiency. Coupling the RS model with an epidemic spread model increased the accuracy (OA = 80%; kappa = 0.48). In the almond trees where the presence of Xf was assayed by qPCR (n = 318 trees), the combined RS-spread model yielded an OA of 71% and kappa = 0.33, which is higher than the RS-only model and visual inspections (both OA = 64-65% and kappa = 0.26-31). Our work demonstrates how combining spatial epidemiological models and remote sensing can lead to highly accurate predictions of plant disease spatial distribution.

Previsual symptoms of Xylella fastidiosa infection revealed in spectral plant-trait alterations.

Plant pathogens cause significant losses to agricultural yields and increasingly threaten food security1, ecosystem integrity and societies in general2-5. Xylella fastidiosa is one of the most dangerous plant bacteria worldwide, causing several diseases with profound impacts on agriculture and the environment6. Primarily occurring in the Americas, its recent discovery in Asia and Europe demonstrates that X. fastidiosa's geographic range has broadened considerably, positioning it as a reemerging global threat that has caused socioeconomic and cultural damage7,8. X. fastidiosa can infect more than 350 plant species worldwide9, and early detection is critical for its eradication8. In this article, we show that changes in plant functional traits retrieved from airborne imaging spectroscopy and thermography can reveal X. fastidiosa infection in olive trees before symptoms are visible. We obtained accuracies of disease detection, confirmed by quantitative polymerase chain reaction, exceeding 80% when high-resolution fluorescence quantified by three-dimensional simulations and thermal stress indicators were coupled with photosynthetic traits sensitive to rapid pigment dynamics and degradation. Moreover, we found that the visually asymptomatic trees originally scored as affected by spectral plant-trait alterations, developed X. fastidiosa symptoms at almost double the rate of the asymptomatic trees classified as not affected by remote sensing. We demonstrate that spectral plant-trait alterations caused by X. fastidiosa infection are detectable previsually at the landscape scale, a critical requirement to help eradicate some of the most devastating plant diseases worldwide.

Assessment of the bacterial community in directly brined Aloreña de Málaga table olive fermentations by metagenetic analysis.

This study uses an "omics" approach to evaluate the bacterial biodiversity changes during fermentation process of natural green cracked Aloreña de Málaga table olives, from raw material to fermented fruit. For this purpose, two industries separated by almost 20km in Guadalhorce Valley (Málaga, Spain) were analysed for obtaining both brines and fruit samples at different moments of fermentation (0, 7, 30 and 120days). Physicochemical and microbial counts during fermentation showed the typical evolution of this type of processes, apparently dominated by yeasts. However, high-throughput barcoded pyrosequencing analysis of V2-V3 hypervariable region of the bacterial 16S rRNA gene showed at 97% identity the presence of 131 bacterial genera included in 357 operational taxonomic units, not detected by the conventional approach. The bacterial biodiversity was clearly higher in the olives at the moment of reception in the industry and during the first days of fermentation, while decreased considerably as elapse the fermentation process. The presence of Enterobacteriaceae and Lactobacillaceae species was scarce during the four months of study. On the contrary, the most important genus at the end of fermentation was Celerinatantimonas in both brine (95.3% of frequency) and fruit (89.4%) samples, while the presence of well-known spoilage microorganisms (Pseudomonas and Propionibacterium) and halophilic bacteria (Modestobacter, Rhodovibrio, Salinibacter) was also common during the course of fermentation. Among the most important bacterial pathogens related to food, only Staphylococcus genus was found at low frequencies (<0.02% of total sequences). Results show the need of this type of studies to enhance our knowledge of the microbiology of table olive fermentations. It is also necessary to determine the role played by these species not previously detected in table olives on the quality and safety of this fermented vegetable.

Soil factors involved in the diversity and structure of soil bacterial communities in commercial organic olive orchards in Southern Spain.

Nowadays, there is a tendency in olive production systems to reduce tillage or keep a vegetative cover to reduce soil erosion and degradation. However, there is scarce information on the effects of different soil management systems (SMS) in soil bacterial community composition of olive groves. In this study, we have evaluated the effects of soil type and different SMS implemented to control weeds in the structure and diversity of bacterial communities of 58 soils in the two geographic areas that best represent the organic olive production systems in Spain. Bacterial community composition assessed by frequency and intensity of occurrence of terminal restriction profiles (TRFs) derived from terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified 16S ribosomal deoxyribonucleic acid were strongly correlated with soil type/field site (Eutric/Calcaric) that differed mainly in soil particle size distribution and soil pH, followed by a strong effect of SMS, in that order. Canonical discriminant (CD) analysis of TRFs properly classified all of the olive orchard soils as belonging to their respective soil type or SMS. Furthermore, only a small set of TRFs were enough to clearly and significantly differentiate soil samples according to soil type or SMS. Those specific TRFs could be used as bioindicators to assess the effect of changes in SMS aimed to enhance soil quality in olive production systems.

In-planta detection and monitorization of endophytic colonization by a Beauveria bassiana strain using a new-developed nested and quantitative PCR-based assay and confocal laser scanning microscopy.

Beauveria bassiana strain 04/01-Tip obtained from larvae of the opium poppy stem gall Iraella luteipes endophytically colonizes opium poppy plants and protect it against this pest. Development of a specific, rapid and sensitive technique that allows accurately determining the process and factors leading to the establishment of this strain in opium poppy plants would be essential to achieve its efficient control in a large field scale. For that purpose in the present study, species-specific primers that can be used in conventional or quantitative PCR protocols were developed for specifically identification and detection of B. bassiana in plant tissues. The combination of the designed BB.fw/BB.rv primer set with the universal ITS1-F/ITS4 primer set in a two-step nested-PCR approach, has allowed the amplification of up to 10fg of B. bassiana. This represented an increase in sensitivity of 10000- and 1000-fold of detection than when using the BB.fw/BB.rv primers in a single or single-tube semi-nested PCR approaches, respectively. The BB.fw and BB.rv primer set were subsequently optimized to be used in real time quantitative PCR assays and allowed to accurately quantify B. bassiana DNA in different plant DNA backgrounds (leaves and seeds) without losing accuracy and efficiency. The qPCR protocol was used to monitor the endophytic colonization of opium poppy leaves byB. bassiana after inoculation with the strain EABb 04/01-Tip, detecting as low as 26fg of target DNA in leaves and a decrease in fungal biomass over time. PCR quantification data were supported in parallel with CLMS by the monitoring of spatial and temporal patterns of leaf and stem colonization using a GFP-tagged transformant of the B. bassiana EABb 04/01-Tip strain, which enabled to demonstrate that B. bassiana effectively colonizes aerial tissues of opium poppy plants mainly through intercellular spaces and even leaf trichomes. A decline in endophytic colonization was also observed by the last sampling times, i.e. from 10 to 15days after inoculation, although fungal structures still remained present in the leaf tissues. These newly developed molecular protocols should facilitate the detection, quantification and monitoring of endophytic B. bassiana strains in different tissues and host plants and would help to unravel the factors and process governing the specific endophytic association between opium poppy and strain EABb 04/01-Tip providing key insights to formulate a sustainable strategy for I. luteipes management in the host.

A comparison of real-time PCR protocols for the quantitative monitoring of asymptomatic olive infections by Verticillium dahliae pathotypes.

Early, specific, and accurate in planta detection and quantification of Verticillium dahliae are essential to prevent the spread of Verticillium wilt in olive using certified pathogen-free planting material and development of resistance. We comparatively assessed the accuracy, specificity, and efficiency of eight real-time quantitative polymerase chain reaction protocols published since 2002 for the specific detection and quantification of V. dahliae in various host plant species and in soil, using a background of DNAs extracted from olive roots, stems, and leaves. Results showed that some of those protocols were not specific for V. dahliae or were inhibited when using backgrounds other than water. Ranking of protocols according to a weighted score system placed protocols TAQ (based on intergenic spacer ribosomal DNA target gene) and SYBR-4 (based on the ß-tubulin 2 target gene) first in sensitivity and efficiency for the quantification of V. dahliae DNA in small amounts and different types of olive tissues (root and stem) tested. Use of TAQ and SYBR-4 protocols allowed accurate quantification of V. dahliae DNA regardless of the background DNA, with a detection limit being fixed at a cycle threshold of 36 (≈18 fg for SYBR-4 and 15 fg for TAQ) of V. dahliae. The amount of DNA from defoliating (D) and nondefoliating (ND) V. dahliae pathotypes was monitored in Verticillium wilt-resistant 'Frantoio' olive using the TAQ and SYBR-4 protocols. In the infection bioassay, higher amounts of D V. dahliae DNA were measured in olive stems, whereas the average amount of fungal DNA in roots was higher for ND-infected plants than D-infected ones. Overall, V. dahliae DNA amounts in all olive tissues tested tended to slightly decrease or remain stable by the end of the experiment (35 days after inoculation). The SYBR-4 and TAQ protocols further enabled detection of V. dahliae in tissues of symptomless plants, suggesting that both techniques can be useful for implementing certification schemes of pathogen-free planting material as well as helpful tools in breeding resistance to V. dahliae in olive.

First Report of Pectobacterium carotovorum Causing Soft Rot of Opium Poppy in Spain.

Opium poppy (Papaver somniferum L.) is an economically important pharmaceutical crop in Spain. Approximately 8,000 ha are cultivated annually in southern and central Spain. To improve yields, opium poppy cultivation is expanding to more humid or irrigated areas of Spain. In the springs of 2005 and 2007, we observed poppy plants with wilt and stem rot symptoms in irrigated, commercial opium poppy (cv. Nigrum) at Carmona and Écija, which are in Seville Province in southern Spain. Closer observations of affected plants revealed darkening and water soaking of the leaves and stem at the soil level, wilting of the lower leaves or the entire plant, and dark brown discoloration of stem vascular tissues and pith of the plant. Severely affected plants became completely rotten and collapsed. Isolations from symptomatic tissues on nutrient agar consistently yielded bacterial colonies. Pure cultures of four representative bacterial strains (two per each of affected field and year of isolation) were used in triplicate for a comparative analysis of biochemical and physiological traits in the 'carotovora' group of Erwinia (1) with known isolates of Pectobacterium carotovorum subsp. carotovorum, P. carotovorum subsp. atrosepticum, and Dickeya chrysanthemi. The isolates from opium poppy were gram negative, facultatively anaerobic, oxidase negative, catalase positive, grew at 37°C, and did not produce gas from D-glucose. Acid was produced from D(+)-arabinose, lactose, and D(+)-trehalose, but not from α-D-methylglucoside. In addition, the opium poppy bacterial isolates caused soft rot on potato slices within 24 h at 25°C and did not induce a hypersensitive reaction on tobacco leaves. Use of the Biolog GN microplates and the OmniLog ID 1.2 system identified the four poppy isolates as P. carotovorum (showing a 66.7% similarity with the subsp. carotovorum). Pathogenicity of poppy isolates was tested on three 6-week-old opium poppy plants (cv. Nigrum) by injecting 100 µl of a bacterial suspension containing 108 CFU/ml in the basal stem. Plants that served as controls were injected with sterile water. Plants were incubated in a growth chamber adjusted to 28°C, 90% relative humidity, and a 14-h photoperiod of fluorescent light of 360 µE·m-2·s-1. Severe symptoms of soft rot and darkening developed on stems of inoculated plants within 3 to 5 days after inoculation. No symptoms developed on control plants. Bacterial strains reisolated from inoculated plants were identified as P. carotovorum on the basis of the Biolog system, as well as biochemical and physiological characters. To our knowledge, this is the first report of P. carotovorum causing soft rot of commercial opium poppy crops in Spain and elsewhere. The presence of this bacterial pathogen to irrigated crops and humid areas may pose an important constraint on the yield of opium poppy crops in Spain. References: (1) R. S. Dickey and A. Kelman. Pages 44-59 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. N. W. Schaad, ed. The American Phytopathological Society, St. Paul, MN, 1988.

Peronospora arborescens Causes Downy Mildew Disease in Commercial Opium Poppy Crops in France.

Opium poppy is a strategic crop for the pharmaceutical industry because it is the only source of morphine, codeine, and thebaine alkaloid drugs. Approximately 7,360 ha (average from 2001 through 2007) of opium poppy (Papaver somniferum) are grown annually in France, mainly in the Northern-East (Champagne-Ardenne) and Centre-West (Centre and Poitou-Charentes) regions of the country. This acreage accounts for nearly 5.6% of the legally cultivated opium poppies worldwide. Disease symptoms resembling those of downy mildew (2) have been observed frequently in those opium-poppy-growing areas, especially in the Charente-Maritime, Cher, Loiret, and Loir et Cher departments. Disease symptoms included chlorotic to light yellow lesions on the leaf blade, curling and thickening of affected tissues, and expanding necrotic lesions that coalesced, eventually giving rise to large necrotic areas or death of the entire leaf tissues and the plant. With wet weather or high relative humidity, sporangiophores with sporangia were produced frequently on the abaxial leaf surface and occasionally on the adaxial side. Peronospora arborescens and P. cristata have been demonstrated as causal agents of opium poppy downy mildew disease and both have been reported in Europe (1-3); however, the specific identity causal agent in commercial opium poppy crops in France has not yet been determined. Microscopic observations of affected leaves in symptomatic opium poppy leaves sampled from three commercial fields in Loiret Department revealed dichotomously branching sporangiophores bearing single sporangia and oospores of shape and measurements similar to those reported for P. arborescens and P. cristata (1,3). Sporangia dimensions of P. arborescens and P. cristata overlapped, making it difficult to differentiate between the two species based solely on morphological characters (3). A species-specific PCR assay protocol (2) that differentiated P. arborescens from P. cristata was used to diagnose the pathogen. Also, the sequence of the complete 5.8S ribosomal DNA gene and internal transcribed spacers (ITS) 1 and 2 were determined and maximum parsimony analysis was performed with the Peronospora spp. data set described by Landa et al. (2). Both species-specific PCR and phylogenetic analyses of ITS sequences showed that P. arborescens was the only Peronospora species associated with the three samples of downy-mildew-affected leaves analyzed. Thus, DNA fragments of 545, 594, and 456 bp were amplified using total DNA extracted from the sampled leaves and P2, P3, and P6 primer pairs (2), respectively. ITS sequences of all three samples showed 100% homology (GenBank Accession No. EU295529). Phylogenetic analyses using Neighbor Joining of those sequences placed the infecting Peronospora sp. in a clade (100% support) that included all P. arborescens sequences from the GenBank database with 99.2 to 99.9% homology among sequences (2,3). To our knowledge, this is the first report and molecular evidence that P. arborescens causes downy mildew disease in commercial opium poppy crops in France. References: (1) S. M. Francis. No. 686 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1981. (2) B. B. Landa et al. Phytopathology 97:1380, 2007. (3) J. B. Scott et al. Phytopathology 93:752, 2003.

First Report of Downy Mildew of Opium Poppy Caused by Peronospora arborescens in Spain.

Opium poppy (Papaver somniferum) is an economically important pharmaceutical crop in Spain with approximately 7,400 ha cultivated annually. In the spring of 2004, severe attacks by a new foliar disease were observed approximately 500 km apart in commercial opium poppy fields in the Castilla-La Mancha and Andalusia regions of central and southern Spain, respectively. The incidence of affected fields ranged from 40 to 50%, and incidence of diseased plants ranged from 20 to 30%. Initial disease symptoms included irregularly shaped, chlorotic-to-light yellow leaf lesions (ranging in size from 0.5 to 4 cm). Affected tissues curled, thickened, and became deformed and necrotic as disease developed. Lesions expanded in size and often coalesced, eventually giving rise to large necrotic areas in leaves or death of entire leaves. In wet weather or conditions of high relative humidity, a dense felt of sporangiophores with sporangia was produced on the abaxial leaf surface and occasionally on the adaxial surface. Microscopic observations revealed sporangiophores branching dichotomically at least four to six times, ending with sterigmata bearing single sporangia. Sporangia were hyaline, elliptical to spherical in shape, and measured 18 to 24 × 14 to 18 µm (average 19 ± 1.2 × 15 ± 1.6 µm). Occasionally, oospores formed in necrotic leaf tissues. Oospores were dark brown (the surface was irregularly ridged) and measured 36 to 46 µm in diameter (average 39 ± 4.4 µm). The oospore wall was 3 to 11 µm thick. On the basis of the observed morphological features of six symptomatic plant samples from fields at Castilla-La Mancha and Andalusia regions, we identified the pathogen as Peronospora arborescens (1). Pathogenicity was confirmed by inoculating 4- to 6-week-old opium poppy plants (cv. nigrum) with an isolate collected from a field in Ecija, Andalusia. Seed of test plants was surface disinfested and germinated under sterile conditions. Plants were sprayed with a suspension of 1 to 5 × 105 sporangia per ml in sterile distilled water. Plants sprayed with sterile water served as controls. There were five replicate plants per treatment. Plants were enclosed in sealed plastic bags and kept in the dark for 24 h. This was followed by incubation in a growth chamber at 21°C, 60 to 90% relative humidity, and a 12-h photoperiod (fluorescent light: 360 µE·m-2·s-1). After 5 to 7 days, typical downy mildew symptoms developed in inoculated plants. All control plants remained symptomless. Sporulation by the pathogen on symptomatic leaves occurred when affected plants were sprayed with water, enclosed in sealed plastic bags, and incubated at 21°C in the dark for 24 h. To our knowledge, this is the first report of P. arborescens infecting opium poppy in Spain. Infestations of poppy weeds (Papaver rhoeas) and wild Papaver somniferum were also observed in affected opium poppy fields, which may bear importance in the epidemiology of the disease as alternative hosts for inoculum increase and survival of P. arborescens under field conditions. References: (1) S. M. Francis. No. 686 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1981.