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Consumers are increasingly demanding higher quality and safety standards for the products they consume, and one of this is wheat flour, the basis of a wide variety of processed products. This major component in the diet of many communities can be contaminated by microorganisms before the grain harvest, or during the grain storage right before processing. These microorganisms include several fungal species, many of which produce mycotoxins, secondary metabolites that can cause severe acute and chronic disorders. Yet, we still know little about the overall composition of fungal communities associated with wheat flour. In this study, we contribute to fill this gap by characterizing the fungal microbiome of different types of wheat flour using culture-dependent and -independent techniques. Qualitatively, these approaches suggested similar results, highlighting the presence of several fungal taxa able to produce mycotoxins. In-vitro isolation of fungal species suggest a higher frequency of Penicillium, while metabarcoding suggest a higher abundance of Alternaria. This discrepancy might reside on the targeted portion of the community (alive vs. overall) or in the specific features of each technique. Thus, this study shows that commercial wheat flour hosts a wide fungal diversity with several taxa potentially representing concerns for consumers, aspects that need more attention throughout the food production chain.

Host resistance is the most practical, long-term, and economically efficient disease control measure for Verticillium wilt in olive caused by the xylem-invading fungus Verticillium dahliae (Vd), and it is at the core of the integrated disease management. Plant’s microbiome at the site of infection may have an influence on the host reaction to pathogens; however, the role of xylem microbial communities in the olive resistance to Vd has been overlooked and remains unexplored to date. This research was focused on elucidating whether in vitro olive propagation may alter the diversity and composition of the xylem-inhabiting microbiome and if those changes may modify the resistance response that a wild olive clone shows to the highly virulent defoliating (D) pathotype of Vd. Results indicated that although there were differences in microbial communities among the different propagation methodologies, most substantial changes occurred when plants were inoculated with Vd, regardless of whether the infection process took place, with a significant increase in the diversity of bacterial communities when the pathogen was present in the soil. Furthermore, it was noticeable that olive plants multiplied under in vitro conditions developed a susceptible reaction to D Vd, characterized by severe wilting symptoms and 100% vascular infection. Moreover, those in vitro propagated plants showed an altered xylem microbiome with a decrease in total OTU numbers as compared to that of plants multiplied under non-aseptic conditions. Overall, 10 keystone bacterial genera were detected in olive xylem regardless of infection by Vd and the propagation procedure of plants (in vitro vs nursery), with Cutibacterium (36.85%), Pseudomonas (20.93%), Anoxybacillus (6.28%), Staphylococcus (4.95%), Methylobacterium-Methylorubrum (3.91%), and Bradyrhizobium (3.54%) being the most abundant. Pseudomonas spp. appeared as the most predominant bacterial group in micropropagated plants and Anoxybacillus appeared as a keystone bacterium in Vd-inoculated plants irrespective of their propagation process. Our results are the first to show a breakdown of resistance to Vd in a wild olive that potentially may be related to a modification of its xylem microbiome and will help to expand our knowledge of the role of indigenous xylem microbiome on host resistance, which can be of use to fight against main vascular diseases of olive., This study was funded by project AGL2016-75606-R (Programa Estatal de I+D Orientado a los Retos de la Sociedad from the Spanish Government, the Spanish State Research Agency, and FEDER-EU). MA-M is a recipient of a research fellowship BES-2017-082361 from the Spanish Ministry of Economy and Competitiveness.

Host resistance is the most practical, long-term and economically efficient disease control measure for Verticillium wilt in olive caused by the xylem-invading fungus Verticillium dahliae (Vd), and it is at the core of the integrated disease management. Plant’s microbiome at the site of infection may have an influence on the host reaction to pathogens; however, the role of xylem microbial communities in the olive resistance to Vd has been overlooked and remain unexplored to date. This research was focused on elucidating whether in vitro olive propagation may alter the diversity and composition of the xylem-inhabiting microbiome and if those changes may modify the resistance response that a wild olive clone shows to the highly virulent defoliating (D) pathotype of Vd. Results indicated that although there were differences in microbial communities among the different propagation methodologies, most substantial changes occurred when plants were inoculated with Vd, regardless whether the infection process took place, with a significant increase in the diversity of bacterial communities when the pathogen was present in the soil. Furthermore, it was noticeable that olive plants multiplied under in vitro conditions developed a susceptible reaction to D Vd, characterized by severe wilting symptoms and 100% vascular infection. Moreover, those in vitro propagated plants showed an altered xylem microbiome with a decrease in total OTU numbers as compared to that of plants multiplied under non-aseptic conditions. Overall, 10 keystone bacterial genera were detected in olive xylem regardless infection by Vd and the propagation procedure of plants (in vitro vs nursery), with Cutibacterium (36.85%), Pseudomonas (20.93%), Anoxybacillus (6.28%), Staphylococcus (4.95%), Methylobacterium-Methylorubrum (3.91%), and Bradyrhizobium (3.54%) being the most abundant. Pseudomonas spp. appeared as the most predominant bacterial group in micropropagated plants and Anoxybacillus appeared as a keystone bacterium in Vd-inoculated plants irrespective of their propagation process. Our results are first to show a breakdown of resistance to Vd in a wild olive that potentially maybe related to a modification of its xylem microbiome, and will help to expand our knowledge of the role of indigenous xylem microbiome on host resistance which can be of use to fight against main vascular diseases of olive.

The astonishing fast development of molecular technologies allows humanity to discover new and exciting relationships in nature and also to understand their complex mechanisms. The growing knowledge on this relationship plant-microorganism has enabled us to describe the beneficial functions of bacteria in terms of plant growth promotion, especially under stress conditions, and on plant defense. Plant root exudates perform a vital function in the establishment, evolution and dynamics of this network, being capable to be used in the recruitment of beneficial bacteria and to form microbial communities in the rhizosphere. The present chapter compile and describe from a molecular point of view, the advances in the understanding of the functions of the root exudates, mainly focused on the two main groups of beneficial bacteria (those implicated in nitrogen-fixation and PGPR) and describing the cutting-edge technologies being implemented just now or in the near future.

Two monoconidial isolates of Trichoderma atroviride Miller and one of Trichoderma virens Giddens and Foster were combined with the contact fungicide fluazinam to improve the in vitro inhibition of Rosellinia necatrix Prillieux and the control of avocado white root rot caused by this fungus. A fluazinam concentration of 0.01 mg L −1 was obtained as the most effective minimum concentration for in vitro combinations of antagonist-fungicide, significantly reducing (on average, by 41%) the growth of a representative collection of pathogen isolates. The combinations of one isolate of Trichoderma atroviride or virens , plus fluazinam at 0.01 mg L −1 showed a greater in vitro growth inhibition of R. necatrix (36 and 65%, respectively) than the use of fluazinam alone (23%). However, the inhibition of R. necatrix growth by the other T. atroviride plus fluazinam was less than 20% and not significantly different from the control plus fluazinam. The isolate of T. virens applied alone or with fluazinam at 0.01 mg L −1 induced growth inhibition (65%) of the pathogen similar to that induced by the control with fluazinam at a concentration of 0.05 mg L −1 . A fluazinam concentration of 0.01% has been confirmed as effective for controlling white root rot of avocado. Furthermore, the combination of fluazinam at 0.001% with Trichoderma improves significantly the effective control of avocado white root rot on infected plants in comparison with treatments with the fungicide or the antagonist alone. In this way, our results suggest that applications of these antagonist/fungicide combinations should be recommended in order to avoid the development of resistance to the fungicide by the pathogen and improve the control of avocado white root rot.

Grapevine trunk diseases (GTDs) are a major threat to the wine and grape industry. The aim of the study was to investigate the antifungal activity against Neofusicoccum parvum, Diplodia seriata, and Botryosphaeria dothidea of &epsilon, polylysine, chitosan oligomers, their conjugates, Streptomyces rochei and S. lavendofoliae culture filtrates, and their binary mixtures with chitosan oligomers. In vitro mycelial growth inhibition tests suggest that the efficacy of these treatments, in particular those based on &epsilon, polylysine and &epsilon, polylysine:chitosan oligomers 1:1 w/w conjugate, against the three Botryosphaeriaceae species would be comparable to or higher than that of conventional synthetic fungicides. In the case of &epsilon, polylysine, EC90 values as low as 227, 26.9, and 22.5 &micro, g&middot, mL&minus, 1 were obtained for N. parvum, D. seriata, and B. dothidea, respectively. Although the efficacy of the conjugate was slightly lower, with EC90 values of 507.5, 580.2, and 497.4 &micro, 1, respectively, it may represent a more cost-effective option to the utilization of pure &epsilon, polylysine. The proposed treatments may offer a viable and sustainable alternative for controlling GTDs.

An increased incidence of dieback from branches in several avocado orchards in southern Spain was observed in 2014. Surveys were conducted from May to October 2014, sampling the affected branches to isolate the causal agents. A total of 68 fungal isolates, recovered from ten avocado orchards, were identified, by morphological characterisation and DNA sequencing, as belonging to the genera: Neofusicoccum parvum (50%), Colletotrichum gloeosporioides (17.6%), Neofusicoccum luteum (16.2%), Neofusicoccum australe (13.2%), Neofusicoccum mediterraneum (1.5%) and Lasiodiplodia theobromae (1.5%). A decreasing level of virulence in artificial inoculations on avocado plants was observed in N. parvum, N. luteum, N. mediterraneum, N. australe, C. gloeosporioides and L. theobromae, there were significant differences among N. parvum and the rest of species of this genus, and significant differences were only observed between N. luteum and C. gloeosporioides. The geographical distribution of N. parvum and N. Luteum covers different areas, while C. gloeosporioides and N. australe are located only in the areas around Benamocarra and Vélez-Málaga (southern Spain), while N. mediterraneum and L. theobromae appear only occasionally. This is the first study of avocado branch cankers in Spain which identifies the causal agents and establishes their pathogenicity groups, with N. parvum as the most important causal agent of avocado dieback in this area., CICE- Junta de Andalucía, Grupo PAIDI, Spain (AGR-235); ERDF funds (EU).

Esca and leaf stripe diseases are two of the most destructive grapevine trunk diseases which are caused by a complex of fungal species, being the Ascomycete Phaeomoniella chlamydospora the most frequently isolated species from affected vines. To date, no curative measures are known for control these diseases; therefore, planting disease-resistant cultivars is a time-tested and sustainable approach for disease management. The aim of this study was to identify sources of resistance to P. chlamydospora among minority and commercial grapevine germplasm collections in Spain. For this purpose, 15 and 38 cultivars from the ITACyL and the EVEGA collections, respectively, were selected based on the visual assessment of symptom expression in standing vines over the last 3 years, and inoculated with P. chlamydospora in a detached cutting assay under greenhouse conditions over two consecutive years. Seven months after inoculation, cuttings were collected and inspected for lesion length. The severity of internal wood symptoms caused by P. chlamydospora varied considerably amongst the cultivars. No foliar symptoms were observed during the experiment. All cultivars were susceptible to fungal necrotic infection, indicating that there is no evidence of qualitative resistance to this Ascomycete fungus., The research was financially supported by The National Institute for Agricultural and Food Research and Technology (INIA) under the project RTA2015-00015-C02-01. David Gramaje and David Ruano-Rosa were supported by the DOC-INIA program from the INIA, co-funded by the European Social Fund. María del Pilar Martínez-Diz was supported by the FPI-INIA program from the INIA. Marcos Andrés-Sodupe was supported by a grant from the Government of La Rioja

The use of biological control agents (BCAs) is of interest within an integrated management strategy of Verticillium wilt of olive (VWO) caused by the soil-borne fungus Verticillium dahliae Kleb. Previous studies have shown that the root/rhizosphere of healthy olive plants is an important reservoir of microorganisms displaying biocontrol activity against VWO (i.e., Pseudomonas strains PICF7 and PIC141). Moreover, these BCAs are already adapted to the ecological niche where they are deployed. Three novel bacteria (strains PIC28, PIC73 and PIC167) from nursery-produced olive plants were in-depth characterized using a previously implemented approach consisting of in situ isolation, in vitro antagonism tests, in planta bioassays, phenotypic and metabolic characterization, genome analyses and in silico identification of traits involved in plant-bacteria interactions, and multi-locus sequence analyses. All strains displayed in vitro growth inhibition of different olive pathogens and biocontrol effectiveness against Verticillium dahliae, with strain PIC73 being the most effective BCA. Strains PIC73 and PIC167 were identified as Paenibacillus polymyxa (Prazmowski) Ash et al. and Paenibacillus terrae Yoon et al., respectively. Strain PIC28 belongs to the Bacillus genus. Some of these Bacillales members showed in vitro compatibility with previously characterized BCAs (Pseudomonas spp. strains) also originating from the olive rhizosphere, paving the way for the future development of tailored bacterial consortia effective against VWO., Supported by grants P12-AGR-667 (Junta de Andalucía) and RECUPERA 2020 (MINECO-CSIC agreement), both co-financed by ERDF from the EU.

The use of biological control agents (BCA), alone or in combination with other management measures, has gained attention over the past decades, driven by the need to seek for sustainable and eco-friendly alternatives to confront plant pathogens. The rhizosphere of olive (Olea europaea L.) plants is a source of bacteria with potential as biocontrol tools against Verticillium wilt of olive (VWO) caused by Verticillium dahliae Kleb. A collection of bacterial isolates from healthy nursery-produced olive (cultivar Picual, susceptible to VWO) plants was generated based on morphological, biochemical and metabolic characteristics, chemical sensitivities, and on their in vitro antagonistic activity against several olive pathogens. Three strains (PIC25, PIC105, and PICF141) showing high in vitro inhibition ability of pathogens' growth, particularly against V. dahliae, were eventually selected. Their effectiveness against VWO caused by the defoliating pathotype of V. dahliae was also demonstrated, strain PICF141 being the rhizobacteria showing the best performance as BCA. Genotypic and phenotypic traits traditionally associated with plant growth promotion and/or biocontrol abilities were evaluated as well (e.g., phytase, xylanase, catalase, cellulase, chitinase, glucanase activities, and siderophore and HCN production). Multi-locus sequence analyses of conserved genes enabled the identification of these strains as Pseudomonas spp. Strain PICF141 was affiliated to the “Pseudomonas mandelii subgroup,” within the “Pseudomonas fluorescens group,” Pseudomonas lini being the closest species. Strains PIC25 and PIC105 were affiliated to the “Pseudomonas aeruginosa group,” Pseudomonas indica being the closest relative. Moreover, we identified P. indica (PIC105) for the first time as a BCA. Genome sequencing and in silico analyses allowed the identification of traits commonly associated with plant-bacteria interactions. Finally, the root colonization ability of these olive rhizobacteria was assessed, providing valuable information for the future development of formulations based on these strains. A set of actions, from rhizosphere isolation to genome analysis, is proposed and discussed for selecting indigenous rhizobacteria as effective BCAs., Supported by grants P12-AGR-667 (Junta de Andalucía) and RECUPERA 2020 (MINECO-CSIC agreement), both cofounded by ERDF from the EU.

The olive fruit fly (OFF), Bactrocera oleae is the most devastating pest affecting olive fruit worldwide. Previous investigations have addressed the fungal microbiome associated with olive drupes or B. oleae, but the impact of the insect on fungal communities of olive fruit remains undescribed. In the present work, the fungal microbiome of olive drupes, infested and non-infested by the OFF, was investigated in four different localities and cultivars. Olive fruit fly infestations caused a general reduction of the fungal diversity, a higher quantity of the total DNA and an increase in taxa that remained unidentified or had unknown roles. The infestations led to imbalanced fungal communities with the growth of taxa that are usually outcompeted. While it was difficult to establish a cause-effect link between fly infestation and specific fungi, it is clear that the fly alters the natural microbial balance, especially the low abundant taxa. On the other hand, the most abundant ones, were not significantly influenced by the insect. In fact, despite the slight variation between the sampling locations, Aureobasidium, Cladosporium, and Alternaria, were the dominant genera, suggesting the existence of a typical olive fungal microbiome.

A homothallic Phytophthora species was found to be consistently associated with a rot of mature fruits of two local cultivars of olive (Olea europaea) in Calabria, southern Italy. The phylogenetic analysis of sequences of the ITS1‐5.8S‐ITS2 region and cox1 gene enabled its identification as a new species of clade 2, with a basal position compared to previously described subclades. The new species is described formally with the epithet Phytophthora oleae, referring to the natural matrix from which it was isolated. A unique combination of molecular and morphological characters clearly separates P. oleae from other already described Phytophthora species. This new species produced semipapillate, occasionally bipapillate, persistent sporangia on simple sympodially branching sporangiophores as well as globose and smooth‐walled oogonia, paragynous antheridia and spherical, plerotic oospores. The pathogenicity of P. oleae was confirmed in inoculation trials on fruits of three olive cultivars, including the two local cultivars from which the pathogen had been isolated.

During the last decades we have witnessed growing public concern on the abuse/misuse of agrochemicals to control plant pathogens. The fact that some relevant phytopathogens (for instance, the soil-borne fungus Verticillium dahliae Kleb.) are very difficult to control by methods alternative to chemical-based products, has urged researchers to seek effective measures within integrated disease management frameworks. Biological control, alone or in combination with other approaches, emerges as one of the most promising alternatives to confront plant pathogens in a sustainable, environment-friendly strategy. Effectiveness of biological control agents (BCA) largely depends on colonization and persistence capabilities in the ecological niches (e.g. root and/or rhizosphere) where their benefits are expected to be deployed. As a consequence, due to BCA-host specificity (or co-adaptation) the search of potential BCAs in their target environments seems an appropriate strategy. This chapter describes the isolation, identification and characterization of indigenous antagonist bacteria from the olive rhizosphere that can be eventually exploited as BCA against relevant pathogens affecting this woody crop, with emphasis on V. dahliae. The approach here implemented could be of interest for other pathosystems involving trees and soil-borne pathogens.

Seven different strains of Trichoderma isolated from avocado roots showed antagonism to Rosellinia necatrix, which is the causal agent of white root rot. We studied these Trichoderma strains on the basis of the secondary metabolites produced in liquid culture. Five different compounds, namely, 6PP (6-pentyl-α-pyrone), Harzianolide (4-hexa-2,4-dienyl-3-(2-hydroxy-propyl)-5H-furan-2-one), T39butenolide (4-hexa-2,4-dienyl-3-(2-oxo-propyl)-5H-furan-2-one), Dehydroharzianolide (4-hexa-2,4-dienyl-3-propenyl-5H-furan-2-one) and Cerinolactone [(3-hydroxy-5-(6-isopropyl-3-methylene-3, 4, 4a, 5, 6, 7, 8, 8a-octahydronaphthalen-2-yl) dihydrofuran-2-one); a recently discovered novel metabolite], were obtained. In vitro studies of the effects of these compounds on different R. necatrix strains isolated from avocado roots and with different virulence demonstrated that 6PP had the strongest effect even at a low concentration. Although unstable, Cerinolactone and T39butenolide also had large effects on R. necatrix, mainly at a concentration of 200 μg. Harzianolide and Dehydroharzianolide exhibited the lowest effects on the pathogen. In vivo studies of Trichoderma metabolites on Lupinus luteus plants demonstrated the delay of white root rot epidemic through preventive application of 6PP or Harzianolide to seeds or plantlets by immersion in solutions of these metabolites at 1 mg l-1 (minimum effective dosage). In contrast, Cerinolactone only was effective at 10 mg l-1 when applied by plantlet immersion. Thus, this study reports the role that these metabolites could play for controlling avocado white root rot caused by R. necatrix. © 2014 Koninklijke Nederlandse Planteziektenkundige Vereniging., This work was supported by the CICEJunta de Andalucía grant (Grupo PAIDI, AGR-235) and by the Spanish Plan Nacional I+D+ I from Ministerio de Ciencia e Innovación (Grant AGL 2008-05453-C02-02/AGR and AGL 2011-030354-CO2-02). In addition, this research was cofinanced by FEDER funds (EU).

Trichoderma harzianum Rifai is a well-known biological control agent (BCA) effective against a wide range of phytopathogens. Since colonization and persistence in the target niche is crucial for biocontrol effectiveness we aimed to: (i) shed light on the olive roots colonization process by T. harzianum CECT 2413, (ii) unravel the fate of its biomass upon application, and (iii) study the in planta interaction with the soil-borne pathogen Verticillium dahliae Kleb. Fluorescently-tagged derivatives of CECT 2413 and V. dahliae and confocal laser scanning microscopy were used. In vitro assays showed for the first time mycoparasitism of V. dahliae by T. harzianum, evidenced by events such as hyphal coiling. In planta assays revealed that CECT 2413 profusely colonized the rhizoplane of olive roots. Interestingly, biomass of the BCA was visualized mainly as chlamydospores. This observation was independent on the presence or absence of the pathogen. Evidence of inner colonization of olive roots by CECT 2413 was not obtained. These results suggest that CECT 2413 is not able to persist in a metabolically-active form when applied as a spore suspension. This may have strong implications in the way this BCA should be introduced and/or formulated to be effective against Verticillium wilt of olive., This work was supported by European Regional Development Fund-cofinanced grants from the Spanish Ministry of Economy and Competitiveness [Project number BIO2012-33904] and ‘Junta de Andalucía’ [Project number AGR-6038].

Fifty-five isolates of Rosellinia necatrix, the cause of common avocado white root rot disease, were collected from south-east Spain and characterised according to their virulence behaviour and their molecular patterns to assess broader levels of genetic diversity. Virulence properties were revealed by in vitro inoculation on avocado plants. Differences in reaction types showed variability among these isolates. No sequence differences were observed when the internal transcribed spacer 1 (ITS1) and ITS2 regions and DNA fragments of the β-tubulin, adenosine triphosphatase and translation elongation factor 1 genes were explored in representive isolates from five virulence groups. Random amplified polymorphic DNA (RAPD) amplifications were also performed for each isolate using 19 random primers. Four of these primers revealed polymorphism among isolates and repetitive and discriminative bands were used to build an unweighted pair group with arithmetic mean tree. However, RAPD clustering showed low stability, and no correlation between RAPD and virulence groups was observed, possibly indicating high levels of sexual recombination.

White root rot, caused by Rosellinia necatrix, is one of the most important diseases in avocado orchards and is particularly widespread on the Mediterranean seaboard of southern Spain. In this study, the presence of the pathogen in soil samples collected from the base of 47 plants showing different symptoms of canopy decline was assessed with a molecular detection method based on real-time Scorpion PCR. Results were compared with symptoms in the canopy and with the traditional method of isolation of R. necatrix from roots and/or bark. The fungus was isolated from 24 samples by the traditional method and from 37 soil samples by the molecular method (cycle threshold values 25·8 to 47·1), demonstrating the higher sensitivity and reliability of the molecular method. A single real-time PCR amplification was sufficient to detect R. necatrix in naturally infested soils. The avoidance of nested PCR has important practical implications because of the reduced costs and risk of cross contamination. Also, it enables faster sample analysis and is more appropriate for quantitative detection. A modified molecular method was also developed to detect R. necatrix in roots and in soils with very low populations of the pathogen.

The expression of antifungal genes from Trichoderma harzianum, mainly chitinases, has been used to confer plant resistance to fungal diseases. However, the biotechnological potential of glucanase genes from Trichoderma has been scarcely assessed. In this research, transgenic strawberry plants expressing the β-1,3-glucanase gene bgn13.1 from T. harzianum, under the control of the CaMV35S promoter, have been generated. After acclimatization, five out of 12 independent lines analysed showed a stunted phenotype when growing in the greenhouse. Moreover, most of the lines displayed a reduced yield due to both a reduction in the number of fruit per plant and a lower fruit size. Several transgenic lines showing higher glucanase activity in leaves than control plants were selected for pathogenicity tests. When inoculated with Colletotrichum acutatum, one of the most important strawberry pathogens, transgenic lines showed lower anthracnose symptoms in leaf and crown than control. In the three lines selected, the percentage of plants showing anthracnose symptoms in crown decreased from 61 % to a mean value of 16.5 %, in control and transgenic lines, respectively. Some transgenic lines also showed an enhanced resistance to Rosellinia necatrix, a soil-borne pathogen causing root and crown rot in strawberry. These results indicate that bgn13.1 from T. harzianum can be used to increase strawberry tolerance to crown rot diseases, although its constitutive expression affects plant growth and fruit yield. Alternative strategies such as the use of tissue specific promoters might avoid the negative effects of bgn13.1 expression in plant performance., This research was supported by the Comisión Interministerial de Ciencia y Tecnología in Spain and FEDER EU Funds (Grant No. AGL2011-30354-C02-01).

This chapter aims to provide an overview of recent efforts devoted to the management of soil-borne pathogens by means of combining organic amendments and biological control agents. This approach constitutes a promising disease control strategy yet insufficiently explored. We first present definitions of fundamental concepts that the reader will find interacting within this control strategy. Then, we offer a summary of some research studies exemplifying this topic, underlying the potential of this approach to manage selected relevant soil-borne diseases. We also present reasons leading researchers, producers, and consumers to focus their attention on environmentally friendly disease management frameworks, as well as strategies for their successful implementation and derived benefits. Finally, we briefly discuss the feasibility of applying combinations of organic amendments and biological control agents to control soil-borne diseases in woody plants.

Goji ( Lycium barbarum ) is a newly introduced crop in Italy. In June 2016, one- to two-year-old goji plants with symptoms of wilt were observed in commercial fields in Calabria, southern Italy. Approximately 10% of plants were affected. …

This study tested the effectiveness of single and combined applications of Trichoderma and rhizobacterial strains to control white root rot (WRR) caused by Rosellinia necatrix in avocado plants. Three Trichoderma, two T. atroviride and one T. virens monoconidal strains and four bacterial strains (Bacillus subtilis, Pseudomonas pseudoalcaligenes and two P. chlororaphis) were assayed to determine their compatibilities in vitro. In addition, the effects of the bacterial filtrates were evaluated against the Trichoderma strains and reciprocally; these filtrates were applied alone or in combination to determine their effectiveness against R. necatrix. Individual control agents or combinations of them were applied to avocado plants that were artificially inoculated with a virulent R. necatrix strain. Compatibility between the combined Trichoderma applications and the bacterial strains was observed and these combinations significantly improved the control of R. necatrix during the in vitro experiments. A relative protective effect of some Trichoderma and bacteria was observed on the control of avocado WRR when they were applied singly. The combinations of T. atroviride strains with bacterial strains P. chlororaphis and P. pseudoalcaligenes showed a better control of avocado WRR, whereas the rest of Trichoderma and bacteria combinations also reduced significantly the level of disease and induced a delay at the onset of disease with respect to avocado plants inoculated either with Trichoderma or bacteria. © 2013 KNPV., This research was supported by the “Consejería de Innovación Ciencia y Empresa”-Junta de Andalucía grant (Grupos PAIDI AGR-169, AGR-235) and by the Plan Nacional I+D+I from Ministerio de Ciencia e Innovación (AGL 2008-05453-C02-01, AGL 2008-05453-C02-02, AGL 2011-030354-CO2-01 and AGL 2011-030354-CO2-02). In addition, this research was co-financed by FEDER funds (EU).

A novel metabolite, 3-hydroxy-5-(6-isopropyl-3-methylene-3,4,4a,5,6,7,8,8a- octahydronaphthalen-2-yl)dihydrofuran-2-one, trivially named cerinolactone (1), has been isolated from culture filtrates of Trichoderma cerinum together with three known butenolides containing the 3,4-dialkylfuran-2(5H)-one nucleus, harzianolide (2), T39butenolide (3), and dehydroharzianolide (4). The structure of 1 was determined by spectroscopic methods, including UV, MS, and 1D and 2D NMR analyses. In vitro tests with the purified compound exhibited activity against Pythium ultimum, Rhizoctonia solani, and Botrytis cinerea., Work by F.V., M.R., S.W., and M.L. has been supported by the following projects: Calabria Region APQ Projects “ S.Re.Va.P- r.O. ” , Mis. 124, Projects “ CA.SVI.PR.OLI. ” and “ MI.P.RE.- VEGE. ” ; PRIN MIUR 2008 - prot. 2008SNPNC2 - prot. 2008WKPAWW; FIRB 2002 prot. RBNE01K2E7; PRIN 2003 prot. 2003070719-003; MIUR PON project no. DD12935 of 02/08/2002; MIUR PON project no. DD1219 of 05/10/2004; MIUR PON project no. DD1801 of 31/12/2004; EU TRICHOEST QLK3-2002-02032; EU 2E-BCA2. Work by I.A.G., D.R.R., and C.L.H. has been supported by the Spanish Plan Nacional I+D+I Grant AGL 2008-05453-C02-02/AGR.

In order to monitor Rosellinia necatrix infection of avocado roots, we generated a plasmid vector (pCPXHY1eGFP) constitutively expressing EGFP and developed a protoplast transformation protocol. Using this protocol, four R. necatrix isolates were efficiently transformed and were shown to stably express EGFP homogeneously while not having any observable effect on pathogenicity. Confocal laser scanning microscopy (CLSM) images of avocado roots infected with the highly virulent isolate CH53-GFP demonstrated that fungal penetration of avocado roots occurs simultaneously at several random sites, but it occurs preferentially in the crown region as well as throughout the lenticels and in the junctions between epidermal cells. Not only were R. necatrix hyphae observed invading the epidermal and cortical root cells, but they were also able to penetrate the primary and secondary xylem. Scanning electron microscopy (SEM) images allowed detailed visualisation of the hyphal network generated by invasion of R. necatrix through the epidermal, cortical and vascular cells, including hyphal anastomosis and branching points. To our knowledge, this is the first report describing the construction of GFP-tagged strains belonging to the genus Rosellinia for monitoring white root rot using CLSM and SEM.

Fifty-six bulk isolates of Trichoderma spp. from avocado (Persea americana Mill.), carnation (Dianthus caryophyllus L.), litchi (Litchi chinensis Sonn), rice (Oryza sativa L.) and sugar beet (Beta vulgaris L.) crops located in southern Spain were evaluated for antagonism against one isolate of Rosellinia necatrix Prill. Isolates of both types of fungi were tested in dual and cellophane culture. The origin, cultural characteristics, overgrowth sporulation and staining of growth medium were recorded. As a result, 21 Trichoderma bulk isolates were selected and their corresponding monoconidial isolates were evaluated as above. Next eight monoconidial Trichoderma isolates with the largest in vitro antagonism were selected, and they were additionally tested against nine representative isolates of R. necatrix from nine virulence groups. These were established after pathogenicity tests on 57 isolates of R. necatrix from diseased avocado orchards in southern Spain. These monoconidial Trichoderma isolates were considered as potential biological control agents with a high potential for effective control of white root rot of avocado. Se analizó el antagonismo de cincuenta y seis aislados masales de Trichoderma spp. procedentes de cultivos de aguacate (Persea americana Mill.), clavel (Dianthus caryophyllus L.), litchi (Litchi chinensis Sonn), arroz (Oryza sativa L.) y remolacha (Beta vulgaris L.) del Sur de España frente a un aislado de Rosellinia necatrix Prill. Los aislados de ambos hongos se analizaron in vitro en cultivos duales y de celofán, valorándose la procedencia, características culturales, sobrecrecimiento, esporulación y tinción del medio de cultivo. Se seleccionaron 21 aislados masales y se evaluaron sus correspondientes monoconídicos mediante los análisis in vitro citados. Posteriormente se seleccionaron los 8 aislados monoconídicos de Trichoderma que presentaron el mayor antagonismo in vitro. Estos 8 aislados se evaluaron adicionalmente mediante análisis frente a nueve aislados representativos de R. necatrix, procedentes de nueve grupos de virulencia. Estos se obtuvieron mediante análisis de patogenicidad de 57 aislados de R. necatrix procedentes de fincas de aguacate enfermas del Sur de España. Aquellos aislados monoconídicos de Trichoderma podrían considerarse como agentes de control biológico con un alto potencial para un efectivo control de la podredumbre blanca del aguacate.