Essential oils from Asteraceae as potential biocontrol tools for tomato pests and diseases

Nowadays, new strategies for pest and disease control to be used in rotation with or replacement of conventional pesticides are required. Essential oils (EOs), as botanical pesticides, provide a potential resource to develop more environmentally friendly and less toxic means of control to be applied in different produces. Tomato crop is affected by many insects and fungal diseases, among which, the insects Trialeurodes vaporariorum and Tuta absoluta, and the fungi Alternaria spp. and Botrytis cinerea are of great incidence. In this work two EOs from Uruguayan specimens of the local species Eupatorium buniifolium and the world-wide distributed Artemisia absinthium (Asteraceae) were characterized in their chemical composition and insecticidal and antifungal activities. We found that the EO from local A. absinthium is rich in oxygenated monoterpenes and belongs to the thujone chemotype (β-Thujone abundance is 56 ± 2 %, and α-Thujone, 1.67 ± 0.07 %). On the other hand, monoterpene hydrocarbons (α-Pinene, 22 ± 2 %) and sesquiterpene hydrocarbons [(E)-β-Guaiene, 10 ± 1 %] are the most abundant components of E. buniifolium EO. Eventhough both EOs chemically differ, they exhibit insecticidal and antifungal activity not only by direct contact but also by contact with their vapors against the tested organisms. These results may indicate that these EOs could be raw material to develop control agents to manage some of the main pests and fungal diseases of tomato crops with only one kind of treatment.     

Interplay between Candida albicans and the Antimicrobial Peptide Armory

Antimicrobial peptides (AMPs) are key elements of innate immunity, which can directly kill multiple bacterial, viral, and fungal pathogens. The medically important fungus Candida albicans colonizes different host niches as part of the normal human microbiota. Proliferation of C. albicans is regulated through a complex balance of host immune defense mechanisms and fungal responses. Expression of AMPs against pathogenic fungi is differentially regulated and initiated by interactions of a variety of fungal pathogen-associated molecular patterns (PAMPs) with pattern recognition receptors (PRRs) on human cells. Inflammatory signaling and other environmental stimuli are also essential to control fungal proliferation and to prevent parasitism. To persist in the host, C. albicans has developed a three-phase AMP evasion strategy, including secretion of peptide effectors, AMP efflux pumps, and regulation of signaling pathways. These mechanisms prevent C. albicans from the antifungal activity of the major AMP classes, including cathelicidins, histatins, and defensins leading to a basal resistance. This minireview summarizes human AMP attack and C. albicans resistance mechanisms and current developments in the use of AMPs as antifungal agents.     

Utilization of agro-industrial orange peel and sugar beet pulp wastes for fungal endo- polygalacturonase production

The pectinase enzymes are involved in several industrial applications, and industrial waste is one of the largest environmental pollutants, so this study aims to Endo-polygalacturonase (endo-PG) producing using Aspergillus niger AUMC 4156, Penicillium oxalicum AUMC 4153 and P. variotii AUMC 4149 by using some agro-industrial wastes (dried orange peel and sugar beet pulp) as a sole raw carbon source for degradation these waste in the process of urban wastes disposal. The fermentation process was carried out as a submerged culture technique under both shaken and static culture conditions. A. niger AUMC 4156 was the most promising producer of endo-PG under static conditions while P. oxalicum AUMC 4153 was the highest producer of endo-PG under shaken conditions. Sugar beet pulp proved to be the most preferable to orange peel as the only source of carbon in both shaken and static cultures. The medium that encompassing orange peel as a single carbon source afforded the highest protein content with all tested fungal strains in stirred and static cultures in comparison with sugar beet pulp. The highest activity of endo-polygalacuronase that produced using A. niger AUMC 4156 and P. oxalicum AUMC 4153 was achieved by using sugar beet pulp at 3% concentration under static cultures, meanwhile maximal enzyme activity produced by both fungal strains required 2% sugar beet pulp under shaken cultures. Sugar beet pulp showed promised potential as a good inducer for endo-polygalacturoase production, and enzymes production depended on fungal strains, culture medium, and submerged fermentation conditions.     

Plant Growth‐promoting Fungus Penicillium oxalicum Enhances Plant Growth and Induces Resistance in Pearl Millet Against Downy Mildew Disease

Plant Growth‐promoting Fungus (PGPF) Penicillium oxalicum was isolated from rhizosphere soil of pearl millet and was tested for its ability to promote growth and induce systemic resistance in pearl millet against downy mildew disease. The fungal isolate P. oxalicum UOM PGPF 16 was identified as P. oxalicum using ITS sequencing and morphological analysis and sequence was deposited at NCBI with accession number KF150220. Pearl millet susceptible seeds were treated with three different inducers (CS, CF and LCF) of PGPF P. oxalicum and all the inducers significantly reduced the downy mildew disease and enhanced plant growth. Among the inducers tested, CS treatment recorded highest seed germination of 91% and 1427 seedling vigour followed by LCF and CF treatments. The vegetative growth parameter and NPK uptake studies under greenhouse conditions revealed that the CS treatment of P. oxalicum remarkably enhanced the parameters tested when compared to control plants. A significant disease protection of 62% and 58% against downy mildew disease was observed in plants pretreated with CS of P. oxalicum under greenhouse and field conditions, respectively. The spatio‐temporal studies revealed that inducers P. oxalicum required a minimum of 3 days for developing maximum disease resistance which was maintained thereafter. The maximum Peroxidase (POX) activity (62.7 U) was observed at 24 h in seedlings treated with CS of PGPF P. oxalicum and the activity gradually reduced at later time points after pathogen inoculation. Chitinase (CHT) activity was significantly higher in inducer treated seedlings when compared to control seedlings inoculated with pathogen after 48 h and remained constant at all time points.     

Bio-protective potential of lactic acid bacteria isolated from fermented wax gourd

The antifungal activities of 85 lactic acid bacteria strains isolated from fermented wax gourd against the four fungal species, Penicillium oxalicum, Aspergillus flavus, Aspergillus sydowii, and Mucor racemosus, were determined. Inhibitory activity against at least one or more fungal species was observed with 27 Weissella cibaria and 11 Weissella paramesenteroides strains. Among these strains, W. cibaria 861006 and W. paramesenteroides 860509 showed greater inhibitory activities and were therefore selected for further analysis. The results suggested that the antifungal activities were originated from the organic acids produced by W. cibaria 861006 and W. paramesenteroides 860509. The application tests indicated that the growth of P. oxalicum could be effectively inhibited by W. cibaria 861006 for 6 days on grape surfaces. However, W. paramesenteroides 860509 could only remain its inhibition effect for 48 h. The findings obtained in this study suggest the potential use of W. cibaria 861006 as a bio-protective agent against fungi for agricultural purposes or ready-to-eat fresh fruit and vegetable products.     

Relationship between number and type of adhesions of Penicillium oxalicum conidia to tomato roots and biological control of tomato wilt

We studied (a) the extent adhesion of Penicillium oxalicum conidia to tomato roots after application of P. oxalicum conidial formulations with or without stickers, (b) the relationship between the extent of conidial adhesion to roots and biocontrol of the conidial formulations against tomato wilt, and (c) colonisation of roots by P. oxalicum. Adhesion of P. oxalicum conidia to tomato roots occurred within the first minute of contact between the root and the conidial formulation and the bonding strength was sufficiently strong to prevent conidial removal from the roots. In addition, some formulations with stickers that increased conidial adhesion to roots improved the biocontrol of tomato wilt, when compared to that of formulations without stickers. A “dried conidia without stickers” with 0.025% Nu-Film 17 had no effect on the biocontrol of tomato wilt, despite good adherence of the conidia to the roots. The numbers of P. oxalicum conidia that adhered to the roots was constant for 60 days after application of a “dried conidia without stickers” conidial formulation. The significance of these results (speed of adhesion, number of adhered conidia, and variability of conidial external surface) are discussed in relation to the biocontrol success of tomato wilt using different types of conidial formulations with and without stickers.     

Biocontrol of Fusarium and Verticillium Wilt of Tomato by Penicillium oxalicum under Greenhouse and Field Conditions

Treatments with conidia of Penicillium oxalicum produced in a solid‐state fermentation system were applied at similar densities (6 × 10⁶ spores/g seedbed substrate) to tomato seedbeds in water suspensions (T1: 5 days before sowing, or T2: 7 days before transplanting; 15 days after sowing), or in mixture with the production substrate (T3: 7 days before transplanting; 15 days after sowing). Treatments T2 and T3 significantly (P = 0.05) reduced fusarium wilt of tomato in both greenhouse (artificial inoculation) (33 and 28%, respectively) and field conditions (naturally infested soils) (51 and 72%, respectively), while treatment T1 was efficient only in greenhouse (52%). Verticillium wilt disease reduction was obtained with T3 in two field experiments (56 and 46%, respectively), while T1 and T2 reduced disease only in one field experiment (52% for both T1 and T2). Treatment with conidia of P. oxalicum plus fermentation substrate (T3) resulted in better establishment of a stable and effective population of P. oxalicum in seedbed soil and rhizosphere providing populations of approx. 10⁷ CFU/g soil before transplanting. Results indicate that it will be necessary to apply P. oxalicum at a rate of approx. 10⁶–10⁷ CFU/g in seedbed substrate and rhizosphere before transplanting for effective control of fusarium and verticillium wilt of tomato, and that formulation of P. oxalicum has a substantial influence on its efficacy.     

Comparison of proteinase inhibitor-inducing activities and phytoalexin elicitor activities of a pure fungal endopolygalacturonase, pectic fragments, and chitosans

Rhizopus stolonifer endopolygalacturonase, an elicitor of casbene synthetase activity in castor bean seedlings, was found to be a potent elicitor of the phytoalexin pisatin in pea pods and of proteinase Inhibitor I in tomato leaves. The enzyme was an active elicitor or inducer only in its active native state; heat-denatured enzyme was inactive in all three systems. The activities of (a) the tomato pectic polysaccharide proteinase inhibitor-inducing factor, (b) a partially acid hydrolyzed proteinase inhibitor-inducing factor, (c) citrus pectic fragments, and (d) chitosan, were also compared in the three bioassay systems. The four oligosaccharide preparations were active in all three systems, but with different degrees of potency. In tomato leaves and pea pods, chitosans were most active, whereas in castor beans, the citrus pectic fragments were the best elicitors. The data presented support the hypothesis that plant and fungal cell wall fragments are important signals in mobilizing a wide variety of biochemically different types of plant defense responses, and that endopolygalacturonases play a key role in releasing the plant cell wall fragments during pest attacks.     

Biofilm Formation and its Impact on Antifungal Therapy

Fungi can protect themselves from host defences and antifungal drugs by the production of an extracellular hydrophobic matrix. Candida biofilms exhibit resistance to antifungal agents from all classes including the azoles, echinocandins, amphotericin B complex, and flucytosine. Although demonstrated on polystyrene and bronchial epithelia cells, until today, only indirect evidence for A. fumigatus biofilms in patients is available. The antifungals with the most activity against biofilms are the liposomal formulation of amphotericin B and agents in the echinocandin drug class. Importantly, echinocandins show excellent anti-biofilm activity against C. albicans at therapeutic concentrations. However, other biofilms formed by moulds, including A. fumigatus, are relatively resistant to echinocandins. Multiple mechanisms contribute to the intrinsic and acquired antifungal resistance during the different stages of fungal biofilm development. During the growth phase of the early biofilm various factors account for biofilm resistance. Combinational and sequential antifungal therapy as well as combination with enhancers can improve the effect of a single drug. Further studies are warranted to develop new therapeutic strategies targeting fungal biofilm-specific resistance mechanisms.     

Genetic engineering strategies for enhancing tomato resistance to fungal and bacterial pathogens

The classification and detailed overview of the currently known effective strategies used to increase the resistance of tomato (Solanum lycopersicum L., syn. Lycopersicon esculentum Mill.) plants to infectious fungal and bacterial diseases by genetic engineering approaches are presented. Modern data on the mechanisms of the protective effect of heterologous genes on the enhancement of transgenic tomato resistance to fungal and bacterial pathogens are discussed.     

Commercialization of antifungal peptides

There remains an urgent and very much unmet medical need for new antifungal therapies. Ideally, the next generation of treatments for nosocomial and community-acquired infections, including those caused by Candida spp, Aspergillus spp, Cryptococcus spp and Fusarium spp, will be more efficacious, with higher therapeutic indices and broader activity spectra than existing antifungal drug classes. Moreover, future antifungal therapeutics should have novel modes of action/drug targets that at least minimise, if not negate, the risk of acquired resistance developing in their target fungal pathogen populations. In short, developing the next generation of antifungals is a tall order and whoever is successful in doing so must address the various and well-described shortcomings of what remains at present, a very limited choice of largely small molecule-based therapeutics against the fungal infection spectrum. Novel peptide antifungals engineered from a template of mammalian, amphibian and even insect endogenous antimicrobial peptides (AMPs) have clear potential to meet these requirements and consequent clinical success in a range of fungal diseases. This potential will hopefully be realised in the future as any number of the promising preclinical candidate antifungal peptides identified to date are developed further towards the clinic. The size of the ever-increasing market potential as well as unmet clinical need for new antifungal treatments is such that succeeding in delivering novel peptide antifungals as safe and potently efficacious therapies for the future will have a significant health-economic impact.     

Commercialization of antifungal peptides

There remains an urgent and very much unmet medical need for new antifungal therapies. Ideally, the next generation of treatments for nosocomial and community-acquired infections, including those caused by Candida spp, Aspergillus spp, Cryptococcus spp and Fusarium spp, will be more efficacious, with higher therapeutic indices and broader activity spectra than existing antifungal drug classes. Moreover, future antifungal therapeutics should have novel modes of action/drug targets that at least minimise, if not negate, the risk of acquired resistance developing in their target fungal pathogen populations. In short, developing the next generation of antifungals is a tall order and whoever is successful in doing so must address the various and well-described shortcomings of what remains at present, a very limited choice of largely small molecule-based therapeutics against the fungal infection spectrum. Novel peptide antifungals engineered from a template of mammalian, amphibian and even insect endogenous antimicrobial peptides (AMPs) have clear potential to meet these requirements and consequent clinical success in a range of fungal diseases. This potential will hopefully be realised in the future as any number of the promising preclinical candidate antifungal peptides identified to date are developed further towards the clinic. The size of the ever-increasing market potential as well as unmet clinical need for new antifungal treatments is such that succeeding in delivering novel peptide antifungals as safe and potently efficacious therapies for the future will have a significant health-economic impact.     

Mentha spicata and Salvia fruticosa composts as soil amendments in tomato cultivation

Τhe potential use of the aromatic plants Mentha spicata L. (spearmint) and Salvia fruticosa Mill. (sage) as soil amendments was evaluated. For this purpose, tomato seeds were sown in pots that had been filled with composts made from these plants and mixed with soil collected from an organically cultivated tomato field. A 2?×?2?×?4 [two types of fertilizer (synthetic and organic), two types of compost (M. spicata and S. fruticosa) and four compost rates (0%, 2%, 4% and 8%, w/w)] factorial experiment was used; the experiment was conducted twice in a growth chamber and lasted 60 days. At 0, 20, 40 and 60 days, after the establishment of the experiment, the soil bacterial and fungal abundance, the growth of nitrifying bacteria, the number of emerging weeds and the shoot length of tomato plants were measured in all treatments; at the end of the experiment, the above and belowground biomass of tomato plants was also determined. Soil microbial density increased with increasing compost rate of both species; the highest fungal and bacterial densities were recorded at 40 and 60 days, after the establishment of the experiment, respectively. Nitrifying bacteria were present in all treatments and at all sampling times. Both composts had a stimulating effect on tomato growth, which was remarkably pronounced with M. spicata. In contrast, weed emergence was reduced, but only in soils amended with M. spicata. The results suggest that M. spicata compost added at a rate of 4% to 8% is a very promising soil amendment, since it stimulates tomato growth, increases soil bacterial and fungal abundance and inhibits weed emergence. Further research is needed to elucidate the mode of action of M. spicata compost, its effect under field conditions and its possible use in mixed crop, rotational crop or cover crop systems.     

Cellular Responses of Candida albicans to Phagocytosis and the Extracellular Activities of Neutrophils Are Critical to Counteract Carbohydrate Starvation,Oxidative and Nitrosative Stress

Neutrophils are key players during Candida albicans infection. However, the relative contributions of neutrophil activities to fungal clearance and the relative importance of the fungal responses that counteract these activities remain unclear. We studied the contributions of the intra- and extracellular antifungal activities of human neutrophils using diagnostic Green Fluorescent Protein (GFP)-marked C. albicans strains. We found that a carbohydrate starvation response, as indicated by up-regulation of glyoxylate cycle genes, was only induced upon phagocytosis of the fungus. Similarly, the nitrosative stress response was only observed in internalised fungal cells. In contrast, the response to oxidative stress was observed in both phagocytosed and non-phagocytosed fungal cells, indicating that oxidative stress is imposed both intra- and extracellularly. We assessed the contributions of carbohydrate starvation, oxidative and nitrosative stress as antifungal activities by analysing the resistance to neutrophil killing of C. albicans mutants lacking key glyoxylate cycle, oxidative and nitrosative stress genes. We found that the glyoxylate cycle plays a crucial role in fungal resistance against neutrophils. The inability to respond to oxidative stress (in cells lacking superoxide dismutase 5 or glutathione reductase 2) renders C. albicans susceptible to neutrophil killing, due to the accumulation of reactive oxygen species (ROS). We also show that neutrophil-derived nitric oxide is crucial for the killing of C. albicans: a yhb1Δ/Δ mutant, unable to detoxify NO^•, was more susceptible to neutrophils, and this phenotype was rescued by the nitric oxide scavenger carboxy-PTIO. The stress responses of C. albicans to neutrophils are partially regulated via the stress regulator Hog1 since a hog1Δ/Δ mutant was clearly less resistant to neutrophils and unable to respond properly to neutrophil-derived attack. Our data indicate that an appropriate fungal response to all three antifungal activities, carbohydrate starvation, nitrosative stress and oxidative stress, is essential for full wild type resistance to neutrophils.     

Diverse Deep-Sea Fungi from the South China Sea and Their Antimicrobial Activity

We investigated the diversity of fungal communities in nine different deep-sea sediment samples of the South China Sea by culture-dependent methods followed by analysis of fungal internal transcribed spacer (ITS) sequences. Although 14 out of 27 identified species were reported in a previous study, 13 species were isolated from sediments of deep-sea environments for the first report. Moreover, these ITS sequences of six isolates shared 84–92 % similarity with their closest matches in GenBank, which suggested that they might be novel phylotypes of genera Ajellomyces, Podosordaria, Torula, and Xylaria. The antimicrobial activities of these fungal isolates were explored using a double-layer technique. A relatively high proportion (56 %) of fungal isolates exhibited antimicrobial activity against at least one pathogenic bacterium or fungus among four marine pathogenic microbes (Micrococcus luteus, Pseudoaltermonas piscida, Aspergerillus versicolor, and A. sydowii). Out of these antimicrobial fungi, the genera Arthrinium, Aspergillus, and Penicillium exhibited antibacterial and antifungal activities, while genus Aureobasidium displayed only antibacterial activity, and genera Acremonium, Cladosporium, Geomyces, and Phaeosphaeriopsis displayed only antifungal activity. To our knowledge, this is the first report to investigate the diversity and antimicrobial activity of culturable deep-sea-derived fungi in the South China Sea. These results suggest that diverse deep-sea fungi from the South China Sea are a potential source for antibiotics’ discovery and further increase the pool of fungi available for natural bioactive product screening.     

Invertebrate models of fungal infection

The morbidity, mortality and economic burden associated with fungal infections, together with the emergence of fungal strains resistant to current antimicrobial agents, necessitate broadening our understanding of fungal pathogenesis and discovering new agents to treat these infections. Using invertebrate hosts, especially the nematode Caenorhabditis elegans and the model insects Drosophila melanogaster and Galleria mellonella, could help achieve these goals. The evolutionary conservation of several aspects of the innate immune response between invertebrates and mammals makes the use of these simple hosts an effective and fast screening method for identifying fungal virulence factors and testing potential antifungal compounds. The purpose of this review is to compare several model hosts that have been used in experimental mycology to-date and to describe their different characteristics and contribution to the study of fungal virulence and the detection of compounds with antifungal properties. This article is part of a Special Issue entitled: Animal Models of Disease.