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Several experiments were carried out to assess the performance of commercial Solanum torvum cultivars against the root knot nematodes Meloidogyne incognitaandM. javanicain Spain. The response ofS. torvum rootstock cultivars Brutus, Espina, Salutamu and Torpedo againstM. incognita andMi-1.2(a) virulentM. javanicaisolates was deter-mined in pot experiments, and of ‘Brutus’ to anN-virulent isolate ofM. incognita, compared with that of the eggplantS. melongena‘Cristal’.

The medical information (MI) function within the pharmaceutical industry plays a significant role in the provision of scientific answers for patients and healthcare professionals. The purpose of this study is to identify the current structure, organization, and functions of MI departments in Spain. Sixty local and international pharmaceutical companies based in Spain were invited to participate in this study between May and July 2019. An online 34–item questionnaire developed by the AMIFE MI Working Group was distributed to the companies. Data were analyzed through descriptive statistics using response frequencies. A total of 44 responses from 60 surveys (73%) were received. More than half of the respondents were employed in international companies based in Europe (57%, n = 25). Seventy-one percent (n = 31) of the companies had 100 to 1,000 employees in Spain. Most respondents declared that they had an MI department in their company (73%, n = 32), with most (53%) having two to five employees working on MI. Most MI (n = 50) specialists had a biomedical academic degree (predominantly pharmacy, biology, and medicine). MI departments were involved in many functions, the most common being answering enquiries (100%), handling drug information requests (97%), preparing written responses (94%), and literature services for external use (77%). The mean annual volume of MI enquiries was 2,301 (median 1,100). The results of this survey contribute to a better understanding of MI departments in Spanish pharmaceutical companies, as well as their functions and responsibilities, and could help identify opportunities and areas for improvement.

Meloidogyne is the most damaging plant parasitic nematode genus affecting vegetable crops worldwide. The induction of plant defense mechanisms against Meloidogyne in tomato by some Trichoderma spp. strains has been proven in pot experiments, but there is no information for tomato bearing the Mi-1.2 resistance gene or for other important fruiting vegetable crops. Moreover, Trichoderma is mostly applied for managing fungal plant pathogens, but there is little information on its effect on nematode-antagonistic fungi naturally occurring in soils. Thus, several experiments were conducted to determine (i) the ability of two commercial formulates of Trichoderma asperellum (T34) and Trichoderma harzianum (T22) to induce systemic resistance in tomato and cucumber against an avirulent Meloidogyne incognita population in split-root experiments; (ii) the effect of combining T34 with tomato carrying the Mi-1.2 resistance gene to an avirulent M. incognita population in sterilized soil; and (iii) the effect of combining T34 with tomato carrying the Mi-1.2 resistance gene to a virulent M. incognita population in two suppressive soils in which Pochonia chlamydosporia is naturally present, and the effect of T34 on the level of P. chlamydosporia egg parasitism. Both Trichoderma formulates induced resistance to M. incognita in tomato but not in cucumber. In tomato, the number of egg masses and eggs per plant were reduced by 71 and 54% by T34, respectively. T22 reduced 48% of the number of eggs per plant but not the number of egg masses. T34 reduced the number of eggs per plant of the virulent M. incognita population in both resistant and susceptible tomato cultivars irrespective of the suppressive soil, and its effect was additive with the Mi-1.2 resistance gene. The percentage of fungal egg parasitism by P. chlamydosporia was not affected by the isolate T34 of T. asperellum.

This research was funded by the Spanish Ministry of Economy and Competitiveness Grant AGL2015-66833-R, with a grant from the Generalitat Valenciana to A. Aranda-Martinez (ACIF/2013/120) as well as a sabbatical grant to L.V. Lopez-Llorca (PR2015-0008).

[EN] The response of two Citrullus amarus accessions, BGV0005164 and BGV0005167, was assessed against different Meloidogyne arenaria, Meloidogyne incognita, and Meloidogyne javanica isolates in pot experiments and against M. incognita in plastic greenhouse. In the pot experiments, plants were inoculated with a second-stage juvenile per cm(3) of sterile sand and maintained in a growth chamber at 25 degrees C for 50 days. The watermelon cv. Sugar Baby was included as a susceptible control for comparison. At the end of the experiments, the number of egg masses and eggs per plant was determined, and the reproduction index was calculated as the percentage of the number of eggs produced in the C. amarus accessions with regard to that produced in the susceptible cv. Sugar Baby. In the plastic greenhouse experiment, the ungrafted watermelon cv. Sugar Baby and watermelons grafted onto each of the C. amarus accessions and onto the watermelon rootstock cv. Robusta were cultivated from May to August 2016 in plots with nematode densities from 46 to 1392 J2 per 250 cm(3) of soil at transplantation. At the end of the experiment, the galling index and the number of eggs per plant were determined, and the reproduction index was calculated. Additionally, the compatibility of the two accessions with the watermelon cv. Sugar Baby and the effect on fruit quality (weight, size, shape, firmness, pH, total soluble solids, and flesh color) were assessed under a hydroponic system in a greenhouse. The commercial rootstocks cv. Cobalt and cv. Robusta were also included. All the Meloidogyne isolates produced less egg masses and eggs per plant on the accessions than on Sugar Baby. Both accessions performed as resistant against M. arenaria, and from highly to moderately resistant to M. incognita and M. javanica in pot experiments. In the plastic greenhouse experiment, both C. amarus accessions performed as resistant to M. incognita. Both C. amarus accessions were compatible with the watermelon cv. Sugar Baby, but only the BGV0005167 accession did not influence the fruit quality. Then, the BGV0005167 accession is a promising rootstock for managing the three tropical root-knot nematode species without influencing watermelon fruit quality., The authors thank MINECO and FEDER for funding projects AGL2013-49040-C2-1-R, AGL2014-53398-C2-2-R-AR, AGL2017-89785-R, and AGL2017-85563-C2-1-R-AR. H. A. Garcia-Mendivil acknowledges CONACYT for suport through a pre-doctoral grant.

[EN] Chitosan is a natural polymer with applications in agriculture, which causes plasma membrane permeabilisation and induction of intracellular reactive oxygen species (ROS) in plants. Chitosan has been mostly applied in the phylloplane to control plant diseases and to enhance plant defences, but has also been considered for controlling root pests. However, the effect of chitosan on roots is virtually unknown. In this work, we show that chitosan interfered with auxin homeostasis in Arabidopsis roots, promoting a 2-3 fold accumulation of indole acetic acid (IAA). We observed chitosan dose-dependent alterations of auxin synthesis, transport and signalling in Arabidopsis roots. As a consequence, high doses of chitosan reduce WOX5 expression in the root apical meristem and arrest root growth. Chitosan also propitiates accumulation of salicylic (SA) and jasmonic (JA) acids in Arabidopsis roots by induction of genes involved in their biosynthesis and signalling. In addition, high-dose chitosan irrigation of tomato and barley plants also arrests root development. Tomato root apices treated with chitosan showed isodiametric cells respect to rectangular cells in the controls. We found that chitosan causes strong alterations in root cell morphology. Our results highlight the importance of considering chitosan dose during agronomical applications to the rhizosphere., This work was supported by AGL 2015 66833-R Grant from the Spanish Ministry of Economy and Competitiveness Grant AGL 2015. We would like to thank Drs Isabel Lopez-Diaz and Esther Carrera for plant hormone quantitation (IBMCP, Valencia, Spain). Part of this work was filed for a patent (P201431399) by L. V. Lopez-Llorca, F. Lopez-Moya and N. Escudero as inventors. We would like to thank Dr Michael Kershaw (University of Exeter) for his English revision and critical comments of the manuscript. We also thank Ms Marta Suarez-Fernandez (University of Alicante) and Mr Alfonso Prieto for their technical support. All the authors reviewed and approved the manuscript.

Citrus tristeza is one of the most destructive citrus diseases and is caused by the phloem-restricted Closterovirus, Citrus tristeza virus. Mild strain CTV-B2 does not cause obvious symptoms on indicators whereas severe strain CTV-B6 causes symptoms, including stem pitting, cupping, yellowing, and stiffening of leaves, and vein corking. Our laboratory has previously characterized changes in transcription in sweet orange separately infected with CTV-B2 and CTV-B6. In the present study, transcriptome analysis of Citrus sinensis in response to double infection by CTV-B2 and CTV-B6 was carried out. Four hundred and eleven transcripts were up-regulated and 356 transcripts were down-regulated prior to the onset of symptoms. Repressed genes were overwhelmingly associated with photosynthesis, and carbon and nucleic acid metabolism. Expression of genes related to the glycolytic, oxidative pentose phosphate (OPP), tricarboxylic acid cycle (TCA) pathways, tetrapyrrole synthesis, redox homeostasis, nucleotide metabolism, protein synthesis and post translational protein modification and folding, and cell organization were all reduced. Ribosomal composition was also greatly altered in response to infection by CTV-B2/CTV-B6. Genes that were induced were related to cell wall structure, secondary and hormone metabolism, responses to biotic stress, regulation of transcription, signaling, and secondary metabolism. Transport systems dedicated to metal ions were especially disturbed and ZIPs (Zinc Transporter Precursors) showed different expression patterns in response to co-infection by CTV-B2/CTV-B6 and single infection by CTV-B2. Host plants experienced root decline that may have contributed to Zn, Fe, and other nutrient deficiencies. Though defense responses, such as, strengthening of the cell wall, alteration of hormone metabolism, secondary metabolites, and signaling pathways, were activated, these defense responses did not suppress the spread of the pathogens and the development of symptoms. The mild strain CTV-B2 did not provide a useful level of cross-protection to citrus against the severe strain CTV-B6.

The nematophagous fungus Pochonia chlamydosporia (Goddard) Zare & Gams is a facultative parasite of nematode females and eggs. This fungus has a world-wide distribution and has the capacity to survive in the absence of the nematodes. Pochonia chlamydosporia is rhizosphere competent and can colonize the rhizosphere of crops of economic importance, such as tomato and barley. The infection of nematode eggs by P. chlamydosporia requires adhesion, differentiation of appressoria, and penetration of their egg shells. Since the 1980s, proteases have been described as the main group of enzymes that the fungus uses to penetrate nematode eggs. Recent studies found that these genes are also expressed when the fungus endophytically colonizes barley roots. The genome of the fungus was sequenced in 2014, this being the first publicly available genome of a fungus parasite of nematode eggs. Since then, many publications based on the fungus genome have been published. Study of the major gene families in the genome shows the tools that the fungus uses in its different lifestyles. Transcriptomic studies using DNA microarrays and RNA-seq have been done to study the molecular mechanisms that regulate endophytic colonization of Pochonia chlamydosporia. A general analysis of the genome allowed us to identify a large number of carbohydrate active enzymes (Cazy) which help the fungus in colonizing plants and infecting nematodes.

Pochonia chlamydosporia (Pc), a nematophagous fungus and root endophyte, uses appressoria and extracellular enzymes, principally proteases, to infect the eggs of plant parasitic nematodes (PPN). Unlike other fungi, Pc is resistant to chitosan, a deacetylated form of chitin, used in agriculture as a biopesticide to control plant pathogens. In the present work, we show that chitosan increases Meloidogyne javanica egg parasitism by P. chlamydosporia. Using antibodies specific to the Pc enzymes VCP1 (a subtilisin), and SCP1 (a serine carboxypeptidase), we demonstrate chitosan elicitation of the fungal proteases during the parasitic process. Chitosan increases VCP1 immuno-labelling in the cell wall of Pc conidia, hyphal tips of germinating spores, and in appressoria on infected M. javanica eggs. These results support the role of proteases in egg parasitism by the fungus and their activation by chitosan. Phylogenetic analysis of the Pc genome reveals a large diversity of subtilisins (S8) and serine carboxypeptidases (S10). The VCP1 group in the S8 tree shows evidence of gene duplication indicating recent adaptations to nutrient sources. Our results demonstrate that chitosan enhances Pc infectivity of nematode eggs through increased proteolytic activities and appressoria formation and might be used to improve the efficacy of M. javanica biocontrol. This research was funded by the Spanish Ministry of Economy and Competitiveness Grant AGL 2011-29297 and with a grant from the University of Alicante to N. Escudero (UAFPU2011). S.R. Ferreira was supported by a doctoral degree fellowship from the CAPES (Brazil).

Description of the work carried out by Sedic, a Spanish library association, for developing a collaborative blog as a communication tool to enable authentic, ongoing conversations over the Net.

· Pochonia chlamydosporia has been intensively studied in nematode control of different crops. We have investigated the interaction between P. chlamydosporia and the model system Arabidopsis thaliana under laboratory conditions in the absence of nematodes. · This study demonstrates that P. chlamydosporia colonizes A. thaliana. Root colonization monitored with green fluorescent protein-tagged P. chlamydosporia and quantitative PCR (qPCR) quantitation methods revealed root cell invasion. Fungal inoculation reduced flowering time and stimulated plant growth, as determined by total FW increase, faster development of inflorescences and siliques, and a higher yield in terms of seed production per plant. · Precocious flowering was associated with significant expression changes in key flowering-time genes. In addition, we also provided molecular and genetic evidence that point towards jasmonate signaling as an important factor to modulate progression of plant colonization by the fungus. · Our results indicate that P. chlamydosporia provides benefits to the plant in addition to its nematophagous activity. This report highlights the potential of P. chlamydosporia to improve yield in economically important crops. This research was funded by grants from the Spanish Ministry of Economy and Competitiveness (AGL2015-66833-R to L.V.LL., and BIO2014-56321-P to A.V. and A.M-L.).

The fungal parasite of nematode eggs Pochonia chlamydosporia is also a root endophyte known to promote growth of some plants. In this study, we analysed the effect of nine P. chlamydosporia isolates from worldwide origin on tomato growth. Experiments were performed at different scales (Petri dish, growth chamber and greenhouse conditions) and developmental stages (seedlings, plantlets and plants). Seven P. chlamydosporia isolates significantly (P < 0.05) increased the number of secondary roots and six of those increased total weight of tomato seedlings. Six P. chlamydosporia isolates also increased root weight of tomato plantlets. Root colonisation varied between different isolates of this fungus. Again P. chlamydosporia significantly increased root growth of tomato plants under greenhouse conditions and reduced flowering and fruiting times (up to 5 and 12 days, respectively) versus uninoculated tomato plants. P. chlamydosporia increased mature fruit weight in tomato plants. The basis of the mechanisms for growth, flowering and yield promotion in tomato by the fungus are unknown. However, we found that P. chlamydosporia can produce Indole-3-acetic acid and solubilise mineral phosphate. These results suggest that plant hormones or nutrient ability could play an important role. Our results put forward the agronomic importance of P. chlamydosporia as biocontrol agent of plant parasitic nematodes with tomato growth promoting capabilities. This research was funded by the Spanish Ministry of Science and Innovation Grants AGL 2008-00716/AGR, AGL 2011–29297 and with a grant from the CONACYT (México) to E. A. Z-G.

A combined chemometrics-metabolomics approach [excitation–emission matrix (EEM) fluorescence spectroscopy, nuclear magnetic resonance (NMR) and high performance liquid chromatography–mass spectrometry (HPLC–MS)] was used to analyse the rhizodeposition of the tritrophic system: tomato, the plant-parasitic nematode Meloidogyne javanica and the nematode-egg parasitic fungus Pochonia chlamydosporia. Exudates from M. javanica roots were sampled at root penetration (early) and gall development (late). EMM indicated that late root exudates from M. javanica treatments contained more aromatic amino acid compounds than the rest (control, P. chlamydosporia or P. chlamydosporia and M. javanica). 1H NMR showed that organic acids (acetate, lactate, malate, succinate and formic acid) and one unassigned aromatic compound (peak no. 22) were the most relevant metabolites in root exudates. Robust principal component analysis (PCA) grouped early exudates for nematode (PC1) or fungus presence (PC3). PCA found (PC1, 73.31 %) increased acetate and reduced lactate and an unassigned peak no. 22 characteristic of M. javanica root exudates resulting from nematode invasion and feeding. An increase of peak no. 22 (PC3, 4.82 %) characteristic of P. chlamydosporia exudates could be a plant “primer” defence. In late ones in PC3 (8.73 %) the presence of the nematode grouped the samples. HPLC–MS determined rhizosphere fingerprints of 16 (early) and 25 (late exudates) m/z signals, respectively. Late signals were exclusive from M. javanica exudates confirming EEM and 1H NMR results. A 235 m/z signal reduced in M. javanica root exudates (early and late) could be a repressed plant defense. This metabolomic approach and other rhizosphere -omics studies could help to improve plant growth and reduce nematode damage sustainably. This research was funded by the Spanish Ministry of Science and Innovation Grants AGL 2008-00716/AGR, AGL 2011-29297 and with a grant from the University of Alicante to N. Escudero (UAFPU2011).

Pochonia chlamydosporia (Pc123) is a fungal parasite of nematode eggs which can colonize endophytically barley and tomato roots. In this paper we use culturing as well as quantitative PCR (qPCR) methods and a stable GFP transformant (Pc123gfp) to analyze the endophytic behavior of the fungus in tomato roots. We found no differences between virulence/root colonization of Pc123 and Pc123gfp on root-knot nematode Meloidogyne javanica eggs and tomato seedlings respectively. Confocal microscopy of Pc123gfp infecting M. javanica eggs revealed details of the process such as penetration hyphae in the egg shell or appressoria and associated post infection hyphae previously unseen. Pc123gfp colonization of tomato roots was low close to the root cap, but increased with the distance to form a patchy hyphal network. Pc123gfp colonized epidermal and cortex tomato root cells and induced plant defenses (papillae). qPCR unlike culturing revealed reduction in fungus root colonization (total and endophytic) with plant development. Pc123gfp was found by qPCR less rhizosphere competent than Pc123. Endophytic colonization by Pc123gfp promoted growth of both roots and shoots of tomato plants vs. uninoculated (control) plants. Tomato roots endophytically colonized by Pc123gfp and inoculated with M. javanica juveniles developed galls and egg masses which were colonized by the fungus. Our results suggest that endophytic colonization of tomato roots by P. chlamydosporia may be relevant for promoting plant growth and perhaps affect managing of root-knot nematode infestations.

Methods The students completed a questionnaire before and after a 2-hour practical lesson about ultrasound. The surveys were divided in three parts: a) questions about theoretical and practical knowledge about ultrasound using a 5-point Likert scale of perceived importance ; b) list of structures and pathologies that the students were able to detect by using the ultrasound; c) open-ended questions and comments. The statisitics are presented as percent means or proportions with p-values from t-testing.