Microbe associated molecular patterns from rhizosphere bacteria trigger germination and Papaver somniferum metabolism under greenhouse conditions.

Ten PGPR from different backgrounds were assayed on Papaver somniferum var. Madrigal to evaluate their potential as biotic elicitors to increase alkaloid content under the rationale that some microbe associated molecular patterns (MAMPs) are able to trigger plant metabolism. First, the 10 strains and their culture media at two different concentrations were tested for their ability to trigger seed germination. Then, the best three strains were tested for their ability to increase seedling growth and alkaloid levels under greenhouse conditions. Only three strains and their culture media enhanced germination. Then, germination enhancing capacity of these best three strains, N5.18 Stenotrophomonas maltophilia, Aur9 Chryseobacterium balustinum and N21.4 Pseudomonas fluorescens was evaluated in soil. Finally, the three strains were applied on seedlings at two time points, by soil drench or by foliar spray. Photosynthesis was measured, plant height was recorded, capsules were weighted and alkaloids analyzed by HPLC. Only N5.18 delivered by foliar spray significantly increased plant height coupled to an increase in total alkaloids and a significant increase in opium poppy straw dry weight; these increases were supported by a better photosynthetic efficiency. The relative contents of morphine, thebaine, codeine and oripavine were affected by this treatment causing a significant increase in morphine coupled to a decrease in thebaine, demonstrating the effectivity of MAMPs from N5.18 in this plant species. Considering the increase in capsule biomass and alkaloids together with the acceleration of germination, strain N5.18 appears as a good candidate to elicit plant metabolism and consequently, to increase productivity of Papaver somniferum.

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.

Systemic protection of Papaver somniferum L. against Iraella luteipes (Hymenoptera: Cynipidae) by an endophytic strain of Beauveria bassiana (Ascomycota: Hypocreales).

The poppy stem gall wasp, Iraella luteipes (Thompson) (Hymenoptera: Cynipidae), is one of the main pests of the opium poppy, Papaver somniferum L., an economically important pharmaceutical crop cultivated worldwide. In a previous study, we obtained from I. luteipes larvae a strain of the entomopathogenic fungus Beauveria bassiana (Ascomycota: Hypocreales) that can become established endophytically in opium poppy plants. A field experiment was conducted to study the ability of this B. bassiana strain to provide systemic protection against damage by I. luteipes in opium poppy in southern Spain for three seasons. Conidial suspensions were applied as seed dressings, leaf sprays, or soil sprays. The effect of the treatment was studied by harvesting fully ripened plants and dissecting I. luteipes larvae from the stem. The effect of treatment on growth and yield was also evaluated. Emergence of I. luteipes adults was not uniform over the 3 yr, with important differences exhibited in the duration of the emergence period, although the flight peaks tended to occur in mid-late April. B. bassiana seed dressings, leaf sprays at the fourth true-leaf stage, and soil sprays were not significantly different in their ability to reduce the number of larvae per plant compared with the controls, with percentage reductions of 36.5-58.5, 64.4-73.4, and 51.9-57.2% in 2005, 2006, and 2007, respectively. Even though the population level of I. luteipes increased over the 3 yr, the efficacy of the fungal inoculation in reducing the larval population was maintained throughout the study period. No significant differences between inoculation methods were detected in the percentage of leaf pieces showing fungal growth when placed on B. bassiana selective medium, with mean values in the range of 10-15% for the three seasons. Leaf pieces from controls did not exhibit any sign of B. bassiana growth when placed on B. bassiana-selective medium. Neither adverse effects on growth and yield nor symptomatic tissues were observed in B. bassiana-treated plants compared with controls in any of the three seasons.

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.