Ten challenges to understand and manage the insect-transmitted, xylem-limited bacterial pathogen Xylella fastidiosa.

An unprecedented plant health emergency in olives has been registered over the last decade in Italy, arguably more severe than what occurred repeatedly in grapes in the US in the last 140 years. These emergencies are epidemics caused by a stealthy pathogen, the xylem-limited, insect-transmitted bacterium Xylella fastidiosa. Although these epidemics spurred research that answered many questions about the biology and management of this pathogen, many gaps in knowledge remain. For this review, we set to represent both the US and European perspectives on the most pressing challenges that need to be addressed. These are presented in ten sections, that we hope will stimulate discussion and interdisciplinary research. We reviewed intrinsic problems that arise from the fastidious growth of X. fastidiosa, the lack of specificity for insect transmission, and the economic and social importance of perennial mature woody plant hosts. Epidemiological models and predictions of pathogen establishment and disease expansion, vital for preparedness, are based on very limited data. Most of the current knowledge has been gathered from a few pathosystems, while several hundred remain to be studied, probably including those that will become the center of the next epidemic. Unfortunately, aspects of a particular pathosystem are not always transferable to others. We recommend diversification of research topics of both fundamental and applied nature addressing multiple pathosystems. To increase preparedness through knowledge acquisition is the best strategy to anticipate and manage diseases caused by this pathogen described as 'the most dangerous plant bacteria known worldwide'.

Two Xylella fastidiosa subsp. multiplex Strains Isolated from Almond in Spain Differ in Plasmid Content and Virulence Traits.

The plant-pathogenic bacterium Xylella fastidiosa is a major threat to agriculture and the environment worldwide. Recent devastating outbreaks in Europe highlight the potential of this pathogen to cause emergent diseases. X. fastidiosa subsp. multiplex ESVL and IVIA5901 strains that belong to sequence type 6 were isolated from almond orchards within the outbreak area in Alicante province (Spain). Both strains share more than 99% of the chromosomal sequences (average nucleotide identity), but the ESVL strain harbors two plasmids (pXF64-Hb_ESVL and pUCLA-ESVL). Here, virulence phenotypes and genome content were compared between both strains, using three strains from the United States as a reference for the phenotypic analyses. Experiments in microfluidic chambers, used as a simulation of xylem vessels, showed that twitching motility was absent in the IVIA5901 strain, whereas the ESVL strain had reduced twitching motility. In general, both Spanish strains had less biofilm formation, less cell aggregation, and lower virulence in tobacco compared with U.S. reference strains. Genome analysis of the two plasmids from ESVL revealed 51 unique coding sequences that were absent in the chromosome of IVIA5901. Comparison of the chromosomes of both strains showed some unique coding sequences and single-nucleotide polymorphisms in each strain, with potential deleterious mutations. Genomic differences found in genes previously associated with adhesion and motility might explain the differences in the phenotypic traits studied. Although additional studies are necessary to infer the potential role of X. fastidiosa plasmids, our results indicate that the presence of plasmids should be considered in the study of the mechanisms of pathogenicity and adaptation in X. fastidiosa to new environments. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Divergent abiotic spectral pathways unravel pathogen stress signals across species.

Plant pathogens pose increasing threats to global food security, causing yield losses that exceed 30% in food-deficit regions. Xylella fastidiosa (Xf) represents the major transboundary plant pest and one of the world's most damaging pathogens in terms of socioeconomic impact. Spectral screening methods are critical to detect non-visual symptoms of early infection and prevent spread. However, the subtle pathogen-induced physiological alterations that are spectrally detectable are entangled with the dynamics of abiotic stresses. Here, using airborne spectroscopy and thermal scanning of areas covering more than one million trees of different species, infections and water stress levels, we reveal the existence of divergent pathogen- and host-specific spectral pathways that can disentangle biotic-induced symptoms. We demonstrate that uncoupling this biotic-abiotic spectral dynamics diminishes the uncertainty in the Xf detection to below 6% across different hosts. Assessing these deviating pathways against another harmful vascular pathogen that produces analogous symptoms, Verticillium dahliae, the divergent routes remained pathogen- and host-specific, revealing detection accuracies exceeding 92% across pathosystems. These urgently needed hyperspectral methods advance early detection of devastating pathogens to reduce the billions in crop losses worldwide.

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.

Evidence that Xylella fastidiosa is the Causal Agent of Almond Leaf Scorch Disease in Alicante, Mainland Spain (Iberian Peninsula).

In 2017, Xylella fastidiosa, a quarantine plant-pathogenic bacterium in Europe, was detected in almond trees associated with leaf scorch symptoms in Alicante, a Mediterranean area in southeastern mainland Spain. The bacterium was detected by serological and molecular techniques, isolated in axenic culture from diseased almond trees, and identified as X. fastidiosa subsp. multiplex sequence type (ST) 6. Inoculation experiments on almond plants in greenhouse trials with a characterized strain of X. fastidiosa subsp. multiplex ST6 isolated in the outbreak area have proved that it was able to multiply and systemically colonize inoculated plants. Disease symptoms characteristic of leaf scorch like those observed in the field were observed in the inoculated almond trees after 1 year. Furthermore, the pathogen was reisolated and identified by molecular tests. With the fulfillment of Koch's postulates, we have demonstrated that X. fastidiosa is the causal agent of the almond leaf scorch disease in the Alicante outbreak.

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.

A new stem nematode, Ditylenchus oncogenus n. sp. (Nematoda: Tylenchida), parasitizing sowthistle from Adriatic coast dunes in southern Italy.

Morphological and molecular analyses of a stem nematode causing a severe disease on infected sowthistle (Sonchus bulbosus) plants, involving the formation of gall-like structures on infected leaves and stems, have led to the description of a new species named Ditylenchus oncogenus n. sp. Morphologically, the new species is characterized by a medium to large body size (all adults more than 1 mm in length); a delicate stylet (9.0-11.0 µm long) with minute, rounded knobs; a long post-vulval uterine sac (c. 65% of the vulva-anus distance); six incisures at the lateral fields and characteristic D. destructor-pattern of spicules (with pronounced ventral tumulus and anteriorly pointed, less sclerotized, cuticle parts present within the lamina). The results of molecular analysis of rRNA gene sequences, including the D2-D3 expansion regions of 28S rRNA, internal transcribed spacer (ITS) rRNA, partial 18S rRNA gene, the protein-coding mitochondrial gene, cytochrome oxidase c subunit I (COI), and the heat-shock protein 90 (hsp90) gene, support the new species status. The results of a host-suitability test indicated that the new species does not parasitize potato (Solanum tuberosum) tubers and broad bean (Vicia faba) seedlings. Histopathological observations on naturally infected sowthistle tissues revealed that D. oncogenus n. sp. causes floral stem neoplasia and midrib leaf gall formation on the type, and to date only known, host. The galls were characterized by extensive hyperplasia, where several necrotic cells in the neoplasic area were directly damaged by feeding of the nematode, whereas a number of adjacent cells showed typical cytological changes, such as granulated cytoplasm with hypertrophied nuclei and nucleoli.

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 Southern Blight of Pepper Caused by Sclerotium rolfsii in Southern Spain.

In May 2009, a stem rot of pepper (Capsicum annuum L.) occurred in a 20-ha field in Hacienda de Tarazona, Seville, in southern Spain. Affected plants appeared singly or were grouped in circular patches as much as 8 to 10 m in diameter. Early symptoms consisted of water-soaked lesions on crown and lower stem tissue in contact with the soil. Plant foliage became pale green and wilted, followed by a complete collapse of the plant. A dense white mycelial mat formed on the lower stem and crown with 1- to 2-mm-diameter, spherical, tan-to-dark brown sclerotia. Lower stem pieces of 12 plants with early disease symptoms were surface sterilized in 0.5% NaOCl, dried, transferred to acidified potato dextrose agar, and incubated at 25 ± 1°C in the dark. Fast-growing fungal colonies with white mycelium and abundant sclerotia developed after 6 to 10 days of incubation. On the basis of morphological characters, the fungus was identified as Sclerotium rolfsii Sacc. (2). To confirm the identity of the pathogen, the ribosomal DNA internal transcribed spacer was amplified and sequenced for two isolates (one of the two exact sequences was deposited as GenBank Accession No. GU080230). The sequence was 99% similar to sequences of Athelia rolfsii (S. rolfsii) in GenBank. Pathogenicity of two isolates was determined by placing two oat seeds colonized by each isolate 0.5 to 1 cm from the stem of 2-week-old pepper plants cv. Cristal (one plant per pot, eight replicates). Plants were incubated in a growth chamber maintained at 28 ± 1°C with a 14-h photoperiod of 360 µE·m-2·s-1 and 60 to 90% relative humidity for 10 days. By the sixth day, discoloration and blight of the foliage and stem was observed. Sclerotia formed around the crown and 88% of the plants died 7 days after inoculation. S. rolfsii was recovered from all affected pepper plants. Noninoculated control plants did not develop symptoms. In southern Spain, S. rolfsii is widely distributed in areas of sugar beet production (1). Because of the wide host range of the pathogen, southern blight could become an important disease of vegetable production in southern Spain. References: (1) R. Jordán-Ramírez et al. IOBC/WPRS Bull. 42:101. 2009. (2) J. E. M. Mordue. CMI Descriptions of Pathogenic Fungi and Bacteria. No. 410, 1974.

First Report of Root-Knot Nematode Meloidogyne hispanica Infecting Grapevines in Southern Spain.

Some commercial vineyards producing the 'Condado de Huelva' wine denomination of origin in Almonte, Bonares, and Rociana (Huelva Province), southern Spain, showed general decline in sandy soils in 2009. Disease surveys revealed severe infections of grapevine rootstock Richter 110 feeder roots and heavy soil infestations by a root-knot nematode (Meloidogyne sp.). Infected plants showed a general decline as the only visible aboveground symptom, but when roots were inspected, moderate to small galls on secondary feeder roots were detected. The Meloidogyne sp. population was extracted and quantified from soil and root samples as previously described (1) and identified by the female perineal pattern, esterase (Est) and malate dehydrogenase (Mdh) phenotypes, and sequencing and maximum parsimony (MP) analysis of the ribosomal DNA region D2-D3 of 28S (2,4). Morphology of the perineal patterns and measurements of the second-stage juveniles (J2s) matched those of the original description of Meloidogyne hispanica (3). Enzyme analysis revealed two slow and a medium Est bands, a strong band, and two additional weaker bands coincident with the S2-M1 and N3 Mdh M. hispanica phenotypes (2,4). D2-D3 sequences of all three populations sampled were 100% homologous (GenBank Accession No. GQ375158). Phylogenetic analyses with MP of those sequences placed the Meloidogyne sp. in a clade (100% support) that included all M. hispanica sequences available from the GenBank database (4). M. hispanica was first found in Seville Province, southern Spain, infecting rootstocks of Prunus spp. Its distribution has been confirmed worldwide on different agricultural crops. Thus, M. hispanica has been reported to be infecting grapevines in South Africa and Australia (4); however, to our knowledge, this is the first report of M. hispanica infecting grapevines in Europe. Our data suggest that M. hispanica may pose a threat for vineyard production in southern Spain since M. hispanica was found in 52.63 and 47.36% of soil and root samples, respectively, from 19 fields in 'Condado de Huelva', with nematode population densities ranging from 2.4 to 129.6 eggs and J2s per 100 cm3 of soil and 1 to 1,797 eggs and J2s per gram of fresh roots. Furthermore, genes that confer resistance to other common root-knot nematodes reported on grapevine in Europe may not protect against M. hispanica. References: (1) K. R. Barker. Nematode extraction and bioassays. Page 19 in: An Advanced Treatise on Meloidogyne. Vol. II, Methodology. K. R. Barker et al., eds. North Carolina State University Graphics, Raleigh, 1985. (2) P. R. Esbenshade and A. C. Triantaphyllou. J. Nematol. 22:10, 1990. (3) H. Hirschmann. J. Nematol. 18:520, 1986. (4) B. B. Landa et al. Plant Dis. 92:1104, 2008.

Spatiotemporal analysis of spread of infections by Verticillium dahliae pathotypes within a high tree density olive orchard in southern Spain.

The development of Verticillium wilt epidemics in olive cv. Arbequina was studied from November 1999 to May 2003 in a drip-irrigated, nontillage orchard established in a soil without a history of the disease at Córdoba, southern Spain. Disease incidence measured at 1-month-intervals increased from 0.2 to 7.8% during this period. Verticillium dahliae infecting the trees was characterized as defoliating (D) or nondefoliating (ND) pathotypes by a specific, multiplex-polymerase chain reaction (PCR) assay. Of the symptomatic trees, 87.2 and 12.8% were infected by the D or ND pathotypes, respectively. Dynamics of disease incidence were described by a generalized logistic model with a multiple sigmoid pattern. In the fitted model, the infection rate was highest in the winter to spring period and decreased to minimum values in the summer to fall period. Binary data of disease incidence was analyzed for point pattern and spatial correlation, either directly or after parsing them in contiguous quadrats. Overall, ordinary runs analysis indicated a departure from randomness of disease within rows. The binomial index of dispersion, interclass correlation, and Taylor's power law for various quadrat sizes suggested aggregation of diseased trees within the quadrat sizes tested. Spatial analysis by distance indices showed a nonrandom arrangement of quadrats containing infected trees. Spatial pattern was characterized by the occurrence of several clusters of infected trees. Increasing clustering over time was generally suggested by stronger values of clustering index over time and by the increase in the size of patch clusters. Significant spatial association was found in the clustering of diseased trees over time across cropping seasons; however, clustering was significant only for infections by D V. dahliae, indicating that infections by the D pathotype were aggregated around initial infections. The number and size of clusters of D V. dahliae-infected trees increased over time. Microsatellite-primed PCR assays of a representative number of V. dahliae isolates from diseased trees indicated that the majority of infecting D isolates shared the fingerprinting profile with D V. dahliae isolated from soil of a naturally infested cotton field in close proximity to the orchard, suggesting that short distance dispersal of the pathogen from this soil to the olive orchard may have occurred.

Infection by Meloidogyne artiellia does not break down resistance to races 0, 1a, and 2 of Fusarium oxysporum f. sp. ciceris in chickpea genotypes.

Fusarium oxysporum f. sp. ciceris, and the root-knot nematode Meloidogyne artiellia, coinfect chickpea crops in several countries of the Mediterranean Basin. The influence of root infection by M. artiellia on the reactions of chickpea genotypes with different reaction to infection with F. oxysporum f. sp. ciceris races 0, 1A, and 2 was investigated under controlled environmental conditions. Results demonstrated that co-infection of chickpea genotypes resistant to specific fungal races by M. artiellia did not influence the Fusarium wilt reaction of the plant, irrespective of the F. oxysporum f. sp. ciceris race assayed. However, in some of the assayed combinations, coinfection by both pathogens significantly affected the level of colonization by the fungus or reproduction of the nematode in the root system. Thus, coinfection of chickpea plants with Foc-0 and M. artiellia significantly decreased the level of colonization of the root system by F. oxysporum f. sp. ciceris in genotypes 'CA 336.14.3.0' and 'PV 61', but not in 'ICC 14216 K' and 'UC 27'. Similarly, the nematode reproduction index was also significantly reduced by coinfection with Foc-0 in the four chickpea genotypes tested and inoculated with this race. Conversely, coinfection of chickpea plants with Foc-1A and M. artiellia significantly increased colonization of the root system by the fungus in all genotypes inoculated with this race, except for line BG 212. Altogether, we confirmed the complete resistance phenotype of 'UC 27' and 'ICC 14216 K' to Foc-0, and of 'ICC 14216 K' to Foc-1A and Foc-2, and demonstrated that this resistance was not modified by coinfection of the resistant plant with M. artiellia.

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.

Role of 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonas spp. in the defense of plant roots.

Plants have evolved strategies of stimulating and supporting specific groups of antagonistic microorganisms in the rhizosphere as a defense against diseases caused by soilborne plant pathogens owing to a lack of genetic resistance to some of the most common and widespread soilborne pathogens. Some of the best examples of natural microbial defense of plant roots occur in disease suppressive soils. Soil suppressiveness against many different diseases has been described. Take-all is an important root disease of wheat, and soils become suppressive to take-all when wheat or barley is grown continuously in a field following a disease outbreak; this phenomenon is known as take-all decline (TAD). In Washington State, USA and The Netherlands, TAD results from the enrichment during monoculture of populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing Pseudomonas fluorescens to a density of 10 (5) CFU/g of root, the threshold required to suppress the take-all pathogen, Gaeumannomyces graminis var. tritici. 2,4-DAPG-producing P. fluorescens also are enriched by monoculture of other crops such as pea and flax, and evidence is accumulating that 2,4-DAPG producers contribute to the defense of plant roots in many different agroecosystems. At this time, 22 distinct genotypes of 2,4-DAPG producers (designated A - T, PfY and PfZ) have been defined by whole-cell repetitive sequence-based (rep)-PCR analysis, restriction fragment length polymorphism (RFLP) analysis of PHLD, and phylogenetic analysis of PHLD, but the number of genotypes is expected to increase. The genotype of an isolate is predictive of its rhizosphere competence on wheat and pea. Multiple genotypes often occur in a single soil and the crop species grown modulates the outcome of the competition among these genotypes in the rhizosphere. 2,4-DAPG producers are highly effective biocontrol agents against a variety of plant diseases and ideally suited for serving as vectors for expressing other biocontrol traits in the rhizosphere.

A New Root-Knot Nematode Parasitizing Sea Rocket from Spanish Mediterranean Coastal Dunes: Meloidogyne dunensis n. sp. (Nematoda: Meloidogynidae).

High infection rates of European sea rocket feeder roots by an unknown root-knot nematode were found in a coastal dune soil at Cullera (Valencia) in central eastern Spain. Morphometry, esterase and malate dehydrogenase electrophoretic phenotypes and phylogenetic trees demonstrated that this nematode species differs clearly from other previously described root-knot nematodes. Studies of host-parasite relationships showed a typical susceptible reaction in naturally infected European sea rocket plants and in artificially inoculated tomato (cv. Roma) and chickpea (cv. UC 27) plants. The species is herein described and illustrated and named as Meloidogyne dunensis n. sp. The new root-knot nematode can be distinguished from other Meloidogyne spp. by: (i) perineal pattern rounded-oval, formed of numerous fine dorsal and ventral cuticle striae and ridges, lateral fields clearly visible; (ii) female excretory pore at the level of stylet knobs, EP/ST ratio 1.6; (iii) second-stage juveniles with hemizonid located 1 to 2 annuli anteriorly to excretory pore and long, narrow, tapering tail; and (iv) males with lateral fields composed of four incisures anteriorly and posteriorly, while six distinct incisures are observed for large part at mid-body. Phylogenetic trees derived from distance and maximum parsimony analyses based on 18S, ITS1-5.8S-ITS2 and D2-D3 of 28S rDNA showed that M. dunensis n. sp. can be differentiated from all described root-knot nematode species, and it is clearly separated from other species with resemblance in morphology, such as M. duytsi, M. maritima, M. mayaguensis and M. minor.

Endophytic colonisation of opium poppy, Papaver somniferum, by an entomopathogenic Beauveria bassiana strain.

Beauveria bassiana strain EABb 04/01-Tip isolated from stem-borer larvae of Timaspis papaveris (Hymenoptera: Cynipidae), a serious pest of opium poppy in Spain, was shown to be able to become established endophytically in this pharmaceutical crop. Microbiological, molecular and light and electron microscopic methods were used to study fungal colonisation and to describe its mode of penetration. After inoculation with a foliar spray of conidia, microbiological methods showed 100% of plants examined 24, 48, 72 and 144 h after treatment to be colonised endophytically by the fungus, although the percentage of previously surface sterilised leaf pieces showing fungal growth was 100% at 24 and 48 h, and 80 and 75% at 72 and 144 h after treatment, respectively. The fungus was also observed in leaf pieces obtained from newly formed leaves, indicating that it could spread from treated leaves to leaves formed after fungal application. For molecular studies, a polymerase chain reaction (PCR) protocol was used to amplify the ITS1-5.8S-ITS2 regions of the rDNA of the plant and the fungus. This procedure allowed the detection of the fungus on the surface of the leaves and also endophytically, but only at 72 h after treatment. A nucleotide BLAST search revealed that the ITS1-5.8S-ITS2 sequence of strain EABb 04/01-Tip showed 100% homology with a similar sequence from Cordyceps bassiana. SEM images revealed that although numerous conidia were observed on the leaf surface, few germinated and penetrated. Intracellular colonisation by B. bassiana was not observed, but hyphae were detected growing into the xylem vessels. The fungus was found to colonise 40.5 +/- 4.3% of seedlings (with two cotyledons and the two first real leaves) from seeds dressed with a fungal spore suspension. These results may have implications in the biological control of T. papaveris, including the possible systemic protection of the plant against this cynipid.