Trunk Injection Delivery of Biocontrol Strains of Trichoderma spp. Effectively Suppresses Nut Rot by Gnomoniopsis castaneae in Chestnut (Castanea sativa Mill.).

Gnomoniopsis castaneae is responsible for brown or chalky nut rot in sweet chestnut (Castanea sativa), causing heavy reductions in nut production. Controlling it is challenging, due to its inconspicuous infections, erratic colonization of host tissues and endophytic lifestyle. Fungicides are not applicable because they are prohibited in chestnut forests and strongly discouraged in fruit chestnut groves. Trichoderma species are safe and wide-spectrum biocontrol agents (BCAs), with a variety of beneficial effects in plant protection. This study tested selected strains of T. viride, T. harzianum and T. atroviride for their ability to suppress G. castaneae. Field experiments were conducted in four chestnut groves (two test plots plus two controls) at two sites with a different microclimate. As the size of the trees were a major drawback for uniform and effective treatments, the Trichoderma strains were delivered directly by trunk injection, using the BITE® (Blade for Infusion in TrEes) endotherapic tool. The BCA application, repeated twice in two subsequent years, significantly reduced nut rot incidence, with a more marked, presumably cumulative, effect in the second year. Our data showed the tested Trichoderma strains retain great potential for the biological control of G. castaneae in chestnut groves. The exploitation of Trichoderma spp. as biopesticides is a novelty in the forestry sector and proves the benefits of these microbes in plant disease protection.

Proliferation and migration of PC-3 prostate cancer cells is counteracted by PPARγ-cladosporol binding-mediated apoptosis and a decreased lipid biosynthesis and accumulation.

OBJECTIVES: Chemoprevention, consisting of the administration of natural and/or synthetic compounds, appears to be an alternative way to common therapeutical approaches to preventing the occurrence of various cancers. Cladosporols, secondary metabolites from Cladosporium tenuissimum, showed a powerful ability in controlling human colon cancer cell proliferation through a peroxisome proliferator-activated receptor gamma (PPARγ)-mediated modulation of gene expression. Hence, we carried out experiments to verify the anticancer properties of cladosporols in human prostate cancer cells. Prostate cancer represents one of the most widespread tumors in which several risk factors play a role in determining its high mortality rate in men. MATERIALS AND METHODS: We assessed, by viability assays, PPARγ silencing and overexpression experiments and western blotting analysis, the anticancer properties of cladosporols in cancer prostate cell lines. RESULTS: Cladosporols A and B selectively inhibited the proliferation of human prostate PNT-1A, LNCaP and PC-3 cells and their most impactful antiproliferative ability towards PC-3 prostate cancer cells, was mediated by PPARγ modulation. Moreover, the anticancer ability of cladosporols implied a sustained apoptosis. Finally, cladosporols negatively regulated the expression of enzymes involved in the biosynthesis of fatty acids and cholesterol, thus enforcing the relationship between prostate cancer development and lipid metabolism dysregulation. CONCLUSION: This is the first work, to our knowledge, in which the role of cladosporols A and B was disclosed in prostate cancer cells. Importantly, the present study highlighted the potential of cladosporols as new therapeutical tools, which, interfering with cell proliferation and lipid pathway dysregulation, may control prostate cancer initiation and progression.

Thousand Cankers Disease in Walnut Trees in Europe: Current Status and Management.

Thousand cankers disease (TCD) is a new deadly disease in walnut trees (Juglans spp.), which is plaguing commercial plantations, natural groves, and ornamental black walnut trees (Juglans nigra) in their native and invasion areas in the US and, more recently, in artificial plantations and amenity trees in the newly-invaded areas in Europe (Italy). This insect/fungus complex arises from the intense trophic activity of the bark beetle vector Pityophthorus juglandis in the phloem of Juglans spp. and the subsequent development of multiple Geosmithia morbida cankers around beetles' entry/exit holes. After an analysis of the main biological and ecological traits of both members of this insect/fungus complex, this review explores the options available for TCD prevention and management. Special focus is given to those diagnostic tools developed for disease detection, surveillance, and monitoring, as well as to existing phytosanitary regulations, protocols, and measures that comply with TCD eradication and containment. Only integrated disease management can effectively curtail the pervasive spread of TCD, thus limiting the damage to natural ecosystems, plantations, and ornamental walnuts.

Ultrastructure of Terpene and Polyphenol Synthesis in the Bark of Cupressus sempervirens After Seiridium cardinale Infection.

Cypress Canker Disease (CCD) pandemic caused by Seiridium cardinale is the major constraint of many Cupressaceae worldwide. One of the main symptoms of the disease is the flow of resin from the cankered barks. While inducible phloem axial resin duct-like structures (PARDs) have recently been characterized from an anatomical point of view, their actual resin production is still being debated and has never been demonstrated. Although the involvement of polyphenolic parenchyma cells (PP cells) in the bark of Cupressus sempervirens after S. cardinale infection was revealed in one of our previous studies using light microscopy, their evolution from the phloem parenchyma cells is yet to be clarified. This study investigated functional and ultrastructural aspects of both PARD-like structures and PP cells by means of more in-depth light (LM) and fluorescence microscopy (FM) combined with histochemical staining (using Sudan red, Fluorol Yellow, NADI Aniline blue black, and Toluidine blue staining), in addition to Transmission Electron Microscope (TEM). Two-year-old stem sections of a C. sempervirens canker-resistant clone (var. "Bolgheri"), artificially inoculated with S. cardinale, were sampled 5, 7, 14, 21, and 45 days after inoculation, for time-course observations. FM observation using Fluorol yellow dye clearly showed the presence of lipid material in PARD-like structures lining cells of the cavity and during their secretion into the duct space/cavity. The same tissues were also positive for NADI staining, revealing the presence of terpenoids. The cytoplasm of the ducts' lining cells was also positive for Sudan red. TEM observation highlighted the involvement of plastids and endoplasmic reticulum in the production of terpenoids and the consequent secretion of terpenoids directly through the plasma membrane, without exhibiting vesicle formation. The presence of a high number of mitochondria around the area of terpenoid production suggests that this process is active and consumes ATP. The LM observations showed that PP cells originated from the phloem parenchyma cells (and possibly albuminous cells) through the accumulation of phenolic substances in the vacuole. Here, plastids were again involved in their production. Thus, the findings of this work suggest that the PARD-like structures can actually be considered PARDs or even bark traumatic resin ducts (BTRD).

Loop-Mediated Isothermal Amplification (LAMP) and SYBR Green qPCR for Fast and Reliable Detection of Geosmithia morbida (Kolarik) in Infected Walnut.

Walnut species (Juglans spp.) are multipurpose trees, widely employed in plantation forestry for high-quality timber and nut production, as well as in urban greening as ornamental plants. These species are currently threatened by the thousand cankers disease (TCD) complex, an insect-fungus association which involves the ascomycete Geosmithia morbida (GM) and its vector, the bark beetle Pityophthorus juglandis. While TCD has been studied extensively where it originated in North America, little research has been carried out in Europe, where it was more recently introduced. A key step in research to cope with this new phytosanitary emergency is the development of effective molecular detection tools. In this work, we report two accurate molecular methods for the diagnosis of GM, based on LAMP (real-time and visual) and SYBR Green qPCR, which are complimentary to and integrated with similar recently developed assays. Our protocols detected GM DNA from pure mycelium and from infected woody tissue with high accuracy, sensitivity, and specificity, without cross-reactivity to a large panel of taxonomically related species. The precision and robustness of our tests guarantee high diagnostic standards and could be used to support field diagnostic end-users in TCD monitoring and surveillance campaigns.

First Documentation of Life Cycle Completion of the Alien Rust Pathogen Melampsoridium hiratsukanum in the Eastern Alps Proves Its Successful Establishment in This Mountain Range.

Melampsoridium hiratsukanum is an alien rust fungus which has spread pervasively throughout several European countries following introduction into North Europe at the end of the 20th century. The authenticity of several records of the Melampsoridium species infecting alder (Alnus spp.) in the northern hemisphere is questionable, due to the misidentification and confusion that surround many of the older reports. Given this complicated taxonomic history, and since a M. hiratsukanum-like rust is strongly impacting Alnus incana stands in the Alps, probably affecting the bank protection role of this species along rivers, the unambiguous identification of this pathogen was a pressing epidemiological and ecological issue. In this study, field surveys, light (LM) and scanning electron microscopy (SEM), and molecular characterization were put together in an attempt to solve the conundrum. Field monitoring data, LM and SEM analyses of key taxonomic traits (length of ostiolar cells of uredinium, uredinio-spore shape and size, spore echinulation, number and position of germ pores) and ITS-rDNA sequence-based identification, convergently and unambiguously connected the rust that is causing the current epidemic to the non-native M. hiratsukanum. We documented the completion of the M. hiratsukanum life cycle on its two taxonomically unrelated broadleaf/conifer hosts. This is the first report of M. hiratsukanum from naturally infected Larix decidua in Europe.

Cladosporols A and B, two natural peroxisome proliferator-activated receptor gamma (PPARγ) agonists, inhibit adipogenesis in 3T3-L1 preadipocytes and cause a conditioned-culture-medium-dependent arrest of HT-29 cell proliferation.

BACKGROUND: Obesity and type 2 diabetes mellitus, which are widespread throughout the world, require therapeutic interventions targeted to solve clinical problems (insulin resistance, hyperglycaemia, dyslipidaemia and steatosis). Several natural compounds are now part of the therapeutic repertoire developed to better manage these pathological conditions. Cladosporols, secondary metabolites from the fungus Cladosporium tenuissimum, have been characterised for their ability to control cell proliferation in human colon cancer cell lines through peroxisome proliferator-activated receptor gamma (PPARγ)-mediated modulation of gene expression. Here, we report data concerning the ability of cladosporols to regulate the differentiation of murine 3T3-L1 preadipocytes. METHODS: Cell counting and MTT assay were used for analysing cell proliferation. RT-PCR and Western blotting assays were performed to evaluate differentiation marker expression. Cell migration was analysed by wound-healing assay. RESULTS: We showed that cladosporol A and B inhibited the storage of lipids in 3T3-L1 mature adipocytes, while their administration did not affect the proliferative ability of preadipocytes. Moreover, both cladosporols downregulated mRNA and protein levels of early (C/EBPα and PPARγ) and late (aP2, LPL, FASN, GLUT-4, adiponectin and leptin) differentiation markers of adipogenesis. Finally, we found that proliferation and migration of HT-29 colorectal cancer cells were inhibited by conditioned medium from cladosporol-treated 3T3-L1 cells compared with the preadipocyte conditioned medium. CONCLUSIONS: To our knowledge, this is the first report describing that cladosporols inhibit in vitro adipogenesis and through this inhibition may interfere with HT-29 cancer cell growth and migration. GENERAL SIGNIFICANCE: Cladosporols are promising tools to inhibit concomitantly adipogenesis and control colon cancer initiation and progression.

Rapid Detection of Pityophthorus juglandis (Blackman) (Coleoptera, Curculionidae) with the Loop-Mediated Isothermal Amplification (LAMP) Method.

The walnut twig beetle Pityophthorus juglandis is a phloem-boring bark beetle responsible, in association with the ascomycete Geosmithia morbida, for the Thousand Cankers Disease (TCD) of walnut trees. The recent finding of TCD in Europe prompted the development of effective diagnostic protocols for the early detection of members of this insect/fungus complex. Here we report the development of a highly efficient, low-cost, and rapid method for detecting the beetle, or even just its biological traces, from environmental samples: the loop-mediated isothermal amplification (LAMP) assay. The method, designed on the 28S ribosomal RNA gene, showed high specificity and sensitivity, with no cross reactivity to other bark beetles and wood-boring insects. The test was successful even with very small amounts of the target insect's nucleic acid, with limit values of 0.64 pg/µL and 3.2 pg/µL for WTB adults and frass, respectively. A comparison of the method (both in real time and visual) with conventional PCR did not display significant differences in terms of LoD. This LAMP protocol will enable quick, low-cost, and early detection of P. juglandis in areas with new infestations and for phytosanitary inspections at vulnerable sites (e.g., seaports, airports, loading stations, storage facilities, and wood processing companies).

Thousand cankers disease in Juglans: Optimizing sampling and identification procedures for the vector Pityophthorus juglandis, and the causal agent Geosmithia morbida.

Lindgren funnel traps were used to monitor Pityophthorus juglandis occurrence. Traps were placed directly on walnut trees, with the top tied to one of the lower branches (about 2m high). An 8-funnel model was used instead of a 4-funnel trap, with the specific pheromone bait positioned between the fourth and the fifth funnel. Traps were customized with a 5mm metal mesh which was placed inside the bottom funnel so that debris (mainly foliage) and larger non-target insects would not end up inside the collecting jar. Geosmithia morbida was isolated from beetle adults, larvae and necrotic woody tissue around beetle galleries. Contaminant-free colonies were subcultured in purity and identified by: a) colony phenotyping [morphology, texture and pigmentation; margin type (regular/irregular; lobed/non-lobed); mycelium compactness; surface bumpiness; growth/temperature relationships]; b) micromorphology: type, morphology and ontogeny of conidiophores, metulae and phialides; conidiogenesis; shape, dimension and pigmentation of conidia; c) DNA fingerprinting.•Our protocol was customized to prevent traps from swinging in the wind and to optimize beetle catches by transversely fixing the bottom of funnel traps to the tree trunk with wooden shafts for stability.•To enhance fungus isolation in purity, a semi-selective Potato Dextrose Agar (PDA) medium, enriched with the antibiotics Ampicillin (Policillin-N) and Rifampicin (Rifamycin), was devised to prevent contamination by Gram-positive and Gram-negative bacteria and by mycobacteria.

A duplex real-time PCR with probe for simultaneous detection of Geosmithia morbida and its vector Pityophthorus juglandis.

The cultivation of walnuts (Juglans sp.) in Europe retains high economic, social, and environmental value. The recent reporting of the Thousand Cankers Disease (TCD) fungus, Geosmithia morbida, and of its vector, Pityophthorus juglandis, in walnut trees in Italy is alarming the whole of Europe. Although Italy is at present the only foothold of the disease outside North America, given the difficulties inherent in traditional identification of both members of this beetle/fungus complex, a rapid and effective protocol for the early detection and identification of TCD organisms is an absolute priority for Europe. Here we report the development of an effective and sensitive molecular tool based on simplex/duplex qPCR assays for the rapid, accurate and highly specific detection of both the bionectriaceous fungal pathogen and its bark-beetle vector. Our assay performed excellently, detecting minute amounts of target DNA without any non-specific amplification. Detection limits from various and heterogeneous matrices were lower than other reported assays. Our molecular protocol could assist in TCD organism interception at entry points, territory monitoring for the early identification and eradication of outbreaks, delineation of quarantine areas, and tracing back TCD entry and dispersal pathways.

The antiproliferative and proapoptotic effects of cladosporols A and B are related to their different binding mode as PPARγ ligands.

Cladosporols are secondary metabolites from Cladosporium tenuissimum characterized for their ability to control cell proliferation. We previously showed that cladosporol A inhibits proliferation of human colon cancer cells through a PPARγ-mediated modulation of gene expression. In this work, we investigated cladosporol B, an oxidate form of cladosporol A, and demonstrate that it is more efficient in inhibiting HT-29 cell proliferation due to a robust G0/G1-phase arrest and p21(waf1/cip1) overexpression. Cladosporol B acts as a PPARγ partial agonist with lower affinity and reduced transactivation potential in transient transfections as compared to the full agonists cladosporol A and rosiglitazone. Site-specific PPARγ mutants and surface plasmon resonance (SPR) experiments confirm these conclusions. Cladosporol B in addition displays a sustained proapoptotic activity also validated by p21(waf1/cip1) expression analysis in the presence of the selective PPARγ inhibitor GW9662. In the DMSO/H2O system, cladosporols A and B are unstable and convert to the ring-opened compounds 2A and 2B. Finally, docking experiments provide the structural basis for full and partial PPARγ agonism of 2A and 2B, respectively. In summary, we report here, for the first time, the structural characteristics of the binding of cladosporols, two natural molecules, to PPARγ. The binding of compound 2B is endowed with a lower transactivation potential, higher antiproliferative and proapoptotic activity than the two full agonists as compound 2A and rosiglitazone (RGZ).

Endemic and Emerging Pathogens Threatening Cork Oak Trees: Management Options for Conserving a Unique Forest Ecosystem.

Cork oak (Quercus suber) forests are economically and culturally intertwined with the inhabitants of the Mediterranean basin and characterize its rural landscape. These forests cover over two million hectares in the western Mediterranean basin and sustain a rich biodiversity of endemisms as well as representing an important source of income derived from cork production. Currently cork oak forests are threatened by several factors including human-mediated disturbances such as poor or inappropriate management practices, adverse environmental conditions (irregular water regime with prolonged drought periods), and attacks of pathogens and pests. All these adverse factors can interact, causing a complex disease commonly known as "oak decline." Despite the numerous investigations carried out so far, decline continues to be the main pathological problem of cork oak forests because of its complex etiology and the resulting difficulties in defining suitable control strategies. An overview of the literature indicates that several pathogenic fungi and oomycota can play a primary role in the etiology of this syndrome. Therefore, the aim of this review is to analyze the recent advances achieved regarding the bio-ecology of the endemic and emerging pathogens that threaten cork oak trees with particular emphasis on the species more directly involved in oak decline. Moreover, the effect of climate change on the host-pathogen interactions, a task fundamental for making useful decisions and managing cork oak forests properly, is considered.

Saprophytic growth of the alder rust fungus Melampsoridium hiratsukanum on artificial media.

The first axenic culture of a free living saprophytic stage of the exotic rust fungus Melampsoridium hiratsukanum is reported. Colonies were obtained from one-celled, dikaryotic urediniospores on eight nutrient media out of twelve. Modified Harvey and Grasham (HG) and Schenk and Hildebrandt (HS) media HG1 and SH1 and their bovine serum albumin (BSA)-enriched derivatives gave abundant mycelial growth, but modified Murashige and Skoog (MS) QMS media and their BSA-enriched modifications performed poorly, colony growth being low on QMS-1 and QMS1+BSA, and nil on QMS-5 and QMS-6, with or without BSA. Colonies initially grew poorly when subcultured for one month in purity, but much better after re-transfer to fresh media later: presumably because only the most exploitative genotypes survived, best able to cope with an uncongenial medium. Stabilised cultures survived, and remained vegetative, but only few reproductive colonies produced spore-like bodies. Though the agarised medium remains an inhospitable environment for this biotrophic parasite, it is shown that non-living media can nevertheless sustain the growth and sporulation of this fungus outside its natural hosts and habitat. Axenic culture promises important advances in basic and applied research on this rust, leading to a better understanding of its nutrition, metabolism, diversity and pathogenicity.

Cladosporol A, a new peroxisome proliferator-activated receptor γ (PPARγ) ligand, inhibits colorectal cancer cells proliferation through ß-catenin/TCF pathway inactivation.

BACKGROUND: Cladosporol A, a secondary metabolite from Cladosporium tenuissimum, exhibits antiproliferative properties in human colorectal cancer cells by modulating the expression of some cell cycle genes (p21(waf1/cip1), cyclin D1). METHODS: PPARγ activation by cladosporol A was studied by overexpression and RNA interference assays. The interactions between PPARγ and Sp1 were investigated by co-immunoprecipitation and ChIp assays. ß-Catenin subcellular distribution and ß-catenin/TCF pathway inactivation were analyzed by western blot and RTqPCR, respectively. Cladosporol A-induced ß-catenin proteasomal degradation was examined in the presence of the specific inhibitor MG132. RESULTS: Cladosporol A inhibits cell growth through upregulation of p21(waf1/cip1) gene expression mediated by Sp1-PPARγ interaction. Exposure of HT-29 cells to cladosporol A causes ß-catenin nuclear export, proteasome degradation and reduced expression of its target genes. Upon treatment, PPARγ also activates E-cadherin gene at the mRNA and protein levels. CONCLUSION: In this work we provide evidence that PPARγ mediates the anti-proliferative action of cladosporol A in colorectal cancer cells. Upon ligand activation, PPARγ interacts with Sp1 and stimulates p21(waf1/cip1) gene transcription. PPARγ activation causes degradation of ß-catenin and inactivation of the downstream target pathway and, in addition, upregulates E-cadherin expression reinforcing cell-cell interactions and a differentiated phenotype. GENERAL SIGNIFICANCE: We elucidated the molecular mechanisms by which PPARγ mediates the anticancer activity of cladosporol A.

Secondary mould metabolites of Cladosporium tenuissimum, a hyperparasite of rust fungi.

Investigation of the extracts of a culture of Cladosporium tenuissimum, a known hyperparasite of several rust fungi, gave rise to the isolation of cladosporols B-E (2-5). Their structure and stereochemistry were elucidated on the basis of 1H and 13C NMR evidence and CD measures. Cladosporols 1-5 were active in inhibiting the urediniospore germination of the bean rust agent Uromyces appendiculatus.

Histological studies on the mycoparasitism of Cladosporium tenuissimum on urediniospores of Uromyces appendiculatus.

Interactions between the mycoparasite Cladosporium tenuissimum and the bean rust Uromyces appendiculatus were studied through light and electron microscopy in vitro at the host-parasite interface. Urediniospore germination decreased on contact with ungerminated C. tenuissimum conidia, possibly due to antibiosis mechanisms. C. tenuissimum grew towards the bean rust spores and coiled around their germ tubes. Penetration of the urediniospores occurred either enzymatically and/or mechanically, through appressorium or infection cushion structures, from which a thin penetrating hypha was generated. Enzyme production by the mycoparasite was suggested by the loosening of the matricial components of the spore wall, which sometimes left chitin fibrils visible. Mycoparasite hyphae grew within the host spore, emptied its content, and emerged profusely forming conidiophores and conidia. C. tenuissimum was able to grow on media containing laminarin, suggesting the ability of producing glucanases, but not when chitin was used as the sole carbon source. Conidia that had been grown on a sugar-rich medium, filtered, and extracted with organic solvents, were found to contain cladosporol and related compounds. Complete control of the bean rust disease was achieved by application of C. tenuissimum culture filtrates but not by conidial suspensions. This is the first report of parasitism by C. tenuissimum on U. appendiculatus. These investigations provide additional observations on a genus besides Melampsora and Cronartium from which this fungus has been isolated and tested to date. The possible role of environmental factors for the exploitation of this organism as a biocontrol agent is also mentioned.