In vitro anti-Pythium insidiosum activity of amorolfine hydrochloride and azithromycin, alone and in combination.

Pythium insidiosum infections have been widely studied in an attempt to develop an effective therapeutic protocol for the treatment of human and animal pythiosis. Several antifungal agents are still prescribed against this oomycete, although they present contradictory results. To evaluate the susceptibility profile and to verify the morphological alterations in P. insidiosum isolates treated with amorolfine hydrochloride and azithromycin, alone or in combination. Susceptibility tests for P. insidiosum isolates (n = 20) against amorolfine hydrochloride (AMR) and azithromycin (AZM) were performed according to Clinical and Laboratory Standards Institutes (CLSI) protocol M38-A2. Combinations of both drugs were evaluated using the checkerboard microdilution method. Additionally, transmission and scanning electron microscopy were performed in order to verify the morphological alterations in P. insidiosum isolates in response to these drugs. All P. insidiosum isolates had a minimum inhibitory concentration (MIC) ranging from 16 to 64 mg/l and 8 to 64 mg/l for amorolfine hydrochloride and azithromycin, respectively. Synergistic interactions between the drugs were not observed, with antagonism in 59.8% of isolates, and indifferent interactions in 36.2%. Electron microscopy showed changes in the surface of P. insidiosum hyphae, disorganization of intracellular organelles, and changes in the plasma membrane and cell wall of oomycetes treated with the drugs. This is the first study to demonstrate in vitro anti-P. insidiosum effect of amorolfine hydrochloride. These results indicate the therapeutic potential of this drug against cutaneous and subcutaneous forms of pythiosis, but further studies are necessary to confirm this potential.

Diversity-oriented synthesis and antifungal activities of novel pimprinine derivative bearing a 1,3,4-oxadiazole-5-thioether moiety.

Based on the strategy of diversity-oriented synthesis and the structures of natural product pimprinine and streptochlorin, two series of novel pimprinine derivatives containing 1,3,4-oxadiazole-5-thioether moieties were efficiently synthesized under the optimized reaction conditions. Biological assays conducted at Syngenta showed the designed derivatives displayed an altered pattern of biological activity, of which 5h was identified as the most promising compound with strong activity against Pythium dissimile and also a broad antifungal spectrum in primary screening. Further structural optimization of pimprinine and streptochlorin derivatives is well under way, aiming to discover synthetic analogues with improved antifungal activity. Two series of novel pimprinine derivatives containing 1,3,4-oxadiazole-5-thioether moieties were efficiently synthesized through diversity-oriented synthesis strategy under the optimized conditions. Biological assays showed the designed derivatives exhibited potential activity.

The Toxic Mechanism of Gliotoxins and Biosynthetic Strategies for Toxicity Prevention.

Gliotoxin is a kind of epipolythiodioxopiperazine derived from different fungi that is characterized by a disulfide bridge. Gliotoxins can be biosynthesized by a gli gene cluster and regulated by a positive GliZ regulator. Gliotoxins show cytotoxic effects via the suppression the function of macrophage immune function, inflammation, antiangiogenesis, DNA damage by ROS production, peroxide damage by the inhibition of various enzymes, and apoptosis through different signal pathways. In the other hand, gliotoxins can also be beneficial with different doses. Low doses of gliotoxin can be used as an antioxidant, in the diagnosis and treatment of HIV, and as an anti-tumor agent in the future. Gliotoxins have also been used in the control of plant pathogens, including Pythium ultimum and Sclerotinia sclerotiorum. Thus, it is important to elucidate the toxic mechanism of gliotoxins. The toxic mechanism of gliotoxins and biosynthetic strategies to reduce the toxicity of gliotoxins and their producing strains are summarized in this review.

Enhancing the Potentiality of Trichoderma harzianum against Pythium Pathogen of Beans Using Chamomile (Matricaria chamomilla, L.) Flower Extract.

Our present study was designed to investigate the role of both Trichoderma harzianum and chamomile (Matricaria chamomilla L.) flower extract in mutual reaction against growth of Pythium ultimum. In vitro, the activity of chamomile extract was found to reduce the radial growth of Pythium ultimum up to 30% compared to the control. Whereas, the radial growth reduction effect of T. harzianum against P. ultimum reached 81.6% after 120 h. Data also showed the productivity of total phenolics and total flavonoids by T. harzianum, was 12.18 and 6.33 mg QE/100 mL culture filtrate, respectively. However, these compounds were determined in chamomile flower extract at concentrations of 75.33 and 24.29 mg QE/100 mL, respectively. The fractionation of aqueous extract of chamomile flower using HPLC provided several polyphenolic compounds such as pyrogallol, myricetin, rosemarinic acid, catechol, p-coumaric acid, benzoic acid, chlorogenic acid and other minor compounds. In vivo, the potentiality of T. harzianum with chamomile flower extract against Pythium pathogen of bean was investigated. Data obtained showed a reduction in the percentage of rotted seed and infected seedling up to 28 and 8%, respectively. Whereas, the survival increased up to 64% compared to other ones. There was also a significant promotion in growth features, total chlorophyll, carotenoids, total polyphenols and flavonoids, polyphenol-oxidase and peroxidase enzymes compared to other ones. To the best of our knowledge, there are no reported studies that included the mutual association of fungus, T. harzianum with the extract taken from the chamomile flower against P. ultimum, either in vitro or in vivo. In conclusion, the application of both T. harzianum and/or M. chamomilla extracts in the control of bean Pythium pathogen showed significant results.

In Vitro Susceptibility of Thai Pythium insidiosum Isolates to Antibacterial Agents.

Human pythiosis is a life-threatening human disease caused by Pythium insidiosum In Thailand, vascular pythiosis is the most common form and carries a mortality rate of 10 to 40%, despite aggressive treatment with radical surgery, antifungal agents, and immunotherapy. Itraconazole and terbinafine have been the mainstay of treatment, until recently, based on case report data showing potential synergistic effects against Brazilian P. insidiosum isolates. However, the synergistic effects of itraconazole and terbinafine against Thai P. insidiosum isolates were not observed. This study tested the in vitro susceptibilities of 27 Thai human P. insidiosum isolates (clade II, n = 17; clade IV, n = 10), 12 Thai environmental P. insidiosum isolates (clade II, n = 4; clade IV, n = 8), and 11 non-Thai animal P. insidiosum isolates (clade I, n = 9; clade II, n = 2) to antibiotics in eight antibacterial classes to evaluate alternative effective treatments. Tetracycline and macrolide antibiotics demonstrated in vitro activity against Thai P. insidiosum isolates, with doxycycline MICs (1 to 16 µg/ml), minocycline MICs (1 to 4 µg/ml), tigecycline MICs (1 to 4 µg/ml), azithromycin MICs (1 to 16 µg/ml), and clarithromycin MICs (0.125 to 8 µg/ml) being the lowest, on average. Synergistic effects of tetracyclines and macrolides were also observed.

Biogenic silver nanoparticles in the treatment of experimental pythiosis Bio-AgNP in pythiosis therapy.

Pythiosis is a rapidly progressing disease that can be lethal to affected individuals due to resistance to available therapeutic protocols. The disease affects mammals, with the largest number of reports in horses and humans. The present study investigated the activity of biogenic silver nanoparticles (bioAgNP) in the treatment of experimental pythiosis. The disease was reproduced in nine female 90-day-old New Zealand rabbits. Animals were divided into three groups: group1 (control, n = 3) daily and topically treated with a nonionized gel-based formulation and 1 ml of sterile distilled water intralesion administered every 48 hours; group 2 (n = 3), daily and topically treated with gel-based formulation containing 1 µg/ml bio-AgNP; group 3 (n = 3), treated with 1 ml bio-AgNP in 1 µg/ml aqueous solution intralesion administered every 48 hours. Animals were treated for 45 days, and the area of subcutaneous lesions was measured every 5 days. Results showed that groups 2 and 3 differed from control group (P < .05) in the lesion area, as well as the amount of hyphae within the lesions. It was observed that lesions of treated animals (groups 2 and 3) did not differ from each other, showing that the application route did not influence the regression of lesions. However, it was observed that one animal from group 2 reached clinical cure at 35 days of treatment. This research is pioneer in the application of nanocomposites for the treatment of experimental pythiosis and showed that bio-AgNP can be powerful allies of integrative medicine and can be included in pythiosis therapeutic protocols.

Knockout of Diguanylate Cyclase Genes in Lysobacter enzymogenes to Improve Production of Antifungal Factor and Increase Its Application in Seed Coating.

Heat-stable antifungal factor (HSAF) is a broad-spectrum antifungal antibiotic produced by the biological control agent, Lysobacter enzymogenes. In our earlier works, we have applied HSAF to effectively control wheat and pear fungal disease. However, a major bottleneck in its practical application is the low HSAF production level; therefore, boosting its production is essential for its wide application. In the past, we find that c-di-GMP, a universal bacterial second messenger, is inhibitory to HSAF production. In this work, we further identified eight active diguanylate cyclases (DGCs) responsible for c-di-GMP synthesis in Lysobacter enzymogenes via both bioinformatics and genetic analyses. We generated a strain lacking seven active DGC genes and found that this DGC-modified strain, OH11LC, produced a higher HSAF amount in a c-di-GMP concentration-dependent manner. Subsequently, by employing OH11LC as the host fermentation strain, we could even produce a much higher HSAF amount (> 200-fold). After improving the HSAF production, we further developed a technique of seed coating method with HSAF, which turned out to be effective in fighting against the maize seed-borne filamentous pathogen, Pythium gramineacola. Overall, via combining strain modification and fermentation optimization, we demonstrated a good example of translating fundamental knowledge of bacterial c-di-GMP signaling into biological control application in which we relieved the inhibitory effect of c-di-GMP on HSAF biosynthesis by deleting a bunch of potentially active L. enzymogenes DGC genes to improve HSAF yield and to expand its usage in antifungal seed coating.

An auxin controls bacterial antibiotics production.

The majority of clinically used antibiotics originate from bacteria. As the need for new antibiotics grows, large-scale genome sequencing and mining approaches are being used to identify novel antibiotics. However, this task is hampered by the fact that many antibiotic biosynthetic clusters are not expressed under laboratory conditions. One strategy to overcome this limitation is the identification of signals that activate the expression of silent biosynthetic pathways. Here, we report the use of high-throughput screening to identify signals that control the biosynthesis of the acetyl-CoA carboxylase inhibitor antibiotic andrimid in the broad-range antibiotic-producing rhizobacterium Serratia plymuthica A153. We reveal that the pathway-specific transcriptional activator AdmX recognizes the auxin indole-3-acetic acid (IAA). IAA binding causes conformational changes in AdmX that result in the inhibition of the expression of the andrimid cluster and the suppression of antibiotic production. We also show that IAA synthesis by pathogenic and beneficial plant-associated bacteria inhibits andrimid production in A153. Because IAA is a signalling molecule that is present across all domains of life, this study highlights the importance of intra- and inter-kingdom signalling in the regulation of antibiotic synthesis. Our discovery unravels, for the first time, an IAA-dependent molecular mechanism for the regulation of antibiotic synthesis.

Human Pythiosis: Emergence of Fungal-Like Organism.

Pythiosis is an emerging infectious disease caused by the aquatic oomycete Pythium insidiosum, a fungal-like organism. It is believed that P. insidiosum's zoospores, its infected form, play major role in pathogenesis. Vascular and ocular infections are the most common clinical manifestation in humans. It is difficult to establish the diagnosis given its relatively rarity and difficulty to distinguish P. insidiosum from other molds. Delay in diagnosis and treatment has been associated with poor outcomes. High index of suspicion is the key, particularly in thalassemia patients with arterial insufficiency and patients with fungal keratitis/endophthalmitis without improvement on antifungal therapy. Tissue culture and zoospore induction remain gold standard for diagnosis; however, DNA-based method should be performed simultaneously. The combination of radical surgery, antifungal agents, and immunotherapy has been recommended. It was previously believed that surgery with negative surgical margins was the essential to survive in vascular pythiosis; however, it was recently found that patients could have residual disease despite documented negative surgical margins as infected clot may be dislodged to proximal arterial sites prior to surgery. Serum ß-D-glucan (BG) has been used to monitor disease response after treatment initiation in vascular pythiosis. A significant decrease in BG levels within 2 weeks after surgery is indicative of the absence of residual infection. Unfortunately, monitoring tools for ocular pythiosis are not yet available. Itraconazole plus terbinafine have generally been used in P. insidiosum-infected patients; however, antibacterial agents, including azithromycin and linezolid, have also been used with favorable outcomes in ocular disease. Recently, azithromycin or clarithromycin plus doxycyclin were used in two relapsed vascular pythiosis patients with good outcomes.

Antimicrobial peptide (AMP) from Zingiber zerumbet rhizomes with inhibitory effect on Pythium myriotylum secretory proteases and zoospore viability.

Protease mediated proteolysis has been widely implicated in virulence of necrotrophic fungal pathogens. This is counteracted in plants by evolving new and effective antimicrobial peptides (AMP) that constitute important components of innate immune system. Peptide extraction from rhizome of Zingiber zerumbet was optimized using ammonium sulphate (50-80% w/v) and acetone (60 and 100% v/v) with maximal protein recovery of 1.2 ± 0.4 mg/g obtained using 100% acetone. Evaluation of inhibitory potential of Z. zerumbet rhizome protein extract to prominent hydrolases of necrotrophic Pythium myriotylum revealed maximal inhibition of proteases (75.8%) compared to other hydrolytic enzymes. Protein was purified by Sephacryl S200HR resin resulting in twofold purification and protease inhibition of 84.4%. Non-reducing polyacrylamide gel electrophoresis (PAGE) of the fractions yielded two bands of 75 kDa and 25 kDa molecular size. Peptide mass fingerprint of the protein bands using matrix assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectroscopy (MS) and subsequent MASCOT searches revealed peptide match to methylesterase from Arabidopsis thaliana (15%) and to hypothetical protein from Oryza sativa (98%) respectively. Further centrifugal filter purification using Amicon Ultra (10,000 MW cut-off) filter, yielded a prominent band of 25 kDa size. Concentration dependent inhibition of zoospore viability by Z. zerumbet AMP designated as ZzAMP was observed with maximal inhibition of 89.5% at 4 µg protein and an IC50 value of 0.59 µg. Studies are of particular relevance in the context of identifying the molecules involved in imparting below ground defense in Z. zerumbet as well in development of AMPs as potential candidate molecules for control of necrotrophic pathogens of agricultural relevance.

Efficacy of Azithromycin and Miltefosine in Experimental Systemic Pythiosis in Immunosuppressed Mice.

We evaluated the efficacy of azithromycin (50 mg/kg, every 12 h [q12h] orally) and miltefosine (25 mg/kg, q24h orally) treatments in an experimental model of vascular/disseminated pythiosis in immunosuppressed mice. Azithromycin was the only treatment able to reduce mortality. The histopathological findings showed acute vascular inflammation, pathogen dissemination, necrotizing myositis, neuritis, and arteritis. The results suggest that azithromycin, but not miltefosine, may have clinical relevance in the treatment of vascular/disseminated pythiosis.

The Repurposed Drug Disulfiram Inhibits Urease and Aldehyde Dehydrogenase and Prevents In Vitro Growth of the Oomycete Pythium insidiosum.

Pythium insidiosum is an oomycete microorganism that causes a life-threatening infectious disease, called pythiosis, in humans and animals. The disease has been increasingly reported worldwide. Conventional antifungal drugs are ineffective against P. insidiosum Treatment of pythiosis requires the extensive removal of infected tissue (i.e., eye and leg), but inadequate surgery and recurrent infection often occur. A more effective treatment is needed for pythiosis patients. Drug repurposing is a promising strategy for the identification of a U.S. Food and Drug Administration-approved drug for the control of P. insidiosum Disulfiram has been approved to treat alcoholism, but it exhibits antimicrobial activity against various pathogens. In this study, we explored whether disulfiram possesses an anti-P. insidiosum activity. A total of 27 P. insidiosum strains, isolated from various hosts and geographic areas, were susceptible to disulfiram in a dose-dependent manner. The MIC range of disulfiram against P. insidiosum (8 to 32 mg/liter) was in line with that of other pathogens. Proteogenomic analysis indicated that several potential targets of disulfiram (i.e., aldehyde dehydrogenase and urease) were present in P. insidiosum By homology modeling and molecular docking, disulfiram can bind the putative aldehyde dehydrogenase and urease of P. insidiosum at low energies (i.e., -6.1 and -4.0 Kcal/mol, respectively). Disulfiram diminished the biochemical activities of these enzymes. In conclusion, disulfiram can inhibit the growth of many pathogenic microorganisms, including P. insidiosum The drug can bind and inactivate multiple proteins of P. insidiosum, which may contribute to its broad antimicrobial property. Drug repurposing of disulfiram could be a new treatment option for pythiosis.

Analysis of xanthyletin and secondary metabolites from Pseudomonas stutzeri ST1302 and Klebsiella pneumoniae ST2501 against Pythium insidiosum.

BACKGROUND: Pythium insidiosum is a member of the oomycetes class of aquatic fungus-like microorganisms. It can infect humans and animals through skin wounds and the eyes, causing pythiosis, an infectious disease with high morbidity and mortality rates. Antifungal agents are ineffective as pythiosis treatments because ergosterol, the target site of most antifungal agents, is not found in the P. insidiosum cytoplasmic membrane. The best choice for treatment is surgical removal of the infected organ. While natural plant products or secretory substances from bacterial flora have exhibited in vitro anti-P. insidiosum activity, their mechanism of action remains unknown. Therefore, this study hypothesized that the mechanism of action could be related to changes in P. insidiosum biochemical composition (such as lipid, carbohydrate, protein or nucleic acid) following exposure to the inhibitory substances. The biochemical composition of P. insidiosum was investigated by Synchrotron radiation-based Fourier-transform infrared (FTIR) microspectroscopy. RESULTS: Fraction No.6 from the crude extract of P. stutzeri ST1302, fraction No.1 from the crude extract of K. pneumoniae ST2501 and xanthyletin were used as anti-P. insidiosum substances, with MFCs at 3.125, 1.57-1.91, 0.003 mg/ml, respectively. The synchrotron FTIR results show that the deconvoluted peak distributions in the amide I, amide II, and mixed regions were significantly different between the treatment and control groups. CONCLUSIONS: Xanthyletin and the secondary metabolites from P. stutzeri ST1302 and K. pneumoniae ST2501 exerted anti-P. insidiosum activity that clearly changed the proteins in P. insidiosum. Further study, including proteomics analysis and in vivo susceptibility testing, should be undertaken to develop a better understanding of the mechanism of anti-P. insidiosum activity.

Screening of antibacterial drugs for antimicrobial activity against Pythium insidiosum.

We tested 25 isolates of Pythium insidiosum to investigate their susceptibility to antibacterial drugs that act through inhibition of protein synthesis or other mechanisms of action. We observed that tetracycline, erythromycin, linezolid, nitrofurantoin, Synercid (quinupristin and dalfopristin), chloramphenicol, clindamycin, cetrimide, and crystal violet had inhibitory activity against P. insidiosum. Those in vitro results suggest that antibacterials that inhibit protein synthesis should be the primary antimicrobials investigated for the treatment of pythiosis in animals and humans.

In vitro anti-Pythium insidiosum activity of biogenic silver nanoparticles.

Pythium insidiosum belongs to the phylum Oomycota. It is capable of infecting mammals causing a serious condition called pythiosis, which affects mainly horses in Brazil and humans in Thailand. The objective of the present study was to verify the in vitro anti-P. insidiosum activity of a biogenic silver nanoparticle (bio-AgNP) formulation. The in vitro assays were evaluated on P. insidiosum isolates (n = 38) following the M38-A2 protocol. Damage to the P. insidiosum hyphae ultrastructure was verified by means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Bio-AgNP inhibition concentrations on P. insidiosum isolates ranged from 0.06 to 0.47 µg/ml. It was observed through SEM that P. insidiosum hyphae treated showed surface roughness, as well as cell walls with multiple retraction areas, loss of continuity, and rupture in some areas. The TEM of treated hyphae did not differentiate organelle structures; also, the cellular wall was rarefied, showing wrinkled and partly ruptured borders. The bio-AgNP evaluated has excellent in vitro anti-P. insidiosum activity. However, further studies on its in vivo action are necessary as so to determine the possibility of its use in the treatment of the disease in affected hosts.

Clinical outcomes in ocular pythiosis patients treated with a combination therapy protocol in Thailand: A prospective study.

Ocular pythiosis is the second most common form of human pythiosis, and the rates of evisceration/enucleation in Thailand are 55-79%. This prospective study was conducted to evaluate treatment outcomes of the combination therapy protocol and the potential use of serum (1→3)-ß-glucan (BG) and Pythium insidiosum-specific antibody (Pi-Ab) as an aid to diagnosis and monitoring of ocular pythiosis. Thirty patients were enrolled in the study and 14 (non-globe salvage) required evisceration/enucleation. The globe salvage group was significantly younger, and first ocular surgeries were performed significantly sooner than in the non-globe salvage group. Serum BG and Pi-Ab levels were similar among the 2 groups over time. In vitro susceptibility testing of antifungal agents revealed relatively high minimum inhibitory concentrations and lack of synergistic effect. Serum BG and Pi-Ab would not be useful in diagnosis and monitoring of ocular pythiosis. Until effective antimicrobial agents are discovered, ocular surgeries are still the mainstay therapy in Thailand.

Genome Sequence and Antifungal Activity of Two Niche-Sharing Pseudomonas protegens Related Strains Isolated from Hydroponics.

This work reports the comparison of the genome sequence and the ability to inhibit fungal growth of two Pseudomonas protegens related strains that were isolated from the same hydroponic culture of lamb's lettuce. The two strains were very similar in their core genome but one strain, Pf4, contained three gene clusters for the production of secondary metabolites, i.e., pyoluteorin (plt), pyrrolnitrin (prn), and rhizoxin (rzx), that were missing in the other strain, Pf11. The difference between the two strains was not due to simple insertion events, but to a relatively complex differentiation focused on the accessory genomes. In dual culture assays, both strains inhibited nearly all tested fungal strains, yet Pf4 exerted a significantly stronger fungal growth inhibition than Pf11. In addition to the differences in the secondary metabolite production associated genes abundance, the genome of Pf4 was more stable, smaller in size and with a lower number of transposons. The preservation of a dynamic equilibrium within natural populations of different strains comprised in the same species but differing in their secondary metabolite repertoire and in their genome stability may be functional to the adaptation to environmental changes.

Convergent Evolution of C239S Mutation in Pythium spp. ß-Tubulin Coincides with Inherent Insensitivity to Ethaboxam and Implications for Other Peronosporalean Oomycetes.

Ethaboxam is a benzamide antioomycete chemical (oomicide) used in corn and soybean seed treatments. Benzamides are hypothesized to bind to ß-tubulin, thus disrupting microtubule assembly. Recently, there have been reports of corn- and soybean-associated oomycetes that are insensitive to ethaboxam despite never having been exposed. Here, we investigate the evolutionary history and molecular mechanism of ethaboxam insensitivity. We tested the sensitivity of 194 isolates representing 83 species across four oomycete genera in the Peronosporalean lineage that were never exposed to ethaboxam. In all, 84% of isolates were sensitive to ethaboxam (effective concentration to reduce optical density at 600 nm by 50% when compared with the nonamended control [EC50] < 5 µg ml-1), whereas 16% were insensitive (EC50 > 11 µg ml-1). Of the insensitive isolates, two different transversion mutations were present in the 239th codon in ß-tubulin within three monophyletic groups of Pythium spp. The transversion mutations lead to the same amino acid change from an ancestral cysteine to serine (C239S), which coincides with ethaboxam insensitivity. In a treated soybean seed virulence assay, disease severity was not reduced on ethaboxam-treated seed for an isolate of Pythium aphanidermatum containing a S239 but was reduced for an isolate of P. irregulare containing a C239. We queried publicly available ß-tubulin sequences from other oomycetes in the Peronosporalean lineage to search for C239S mutations from other species not represented in our collection. This search resulted in other taxa that were either homozygous or heterozygous for C239S, including all available species within the genus Peronospora. Evidence presented herein supports the hypothesis that the convergent evolution of C239S within Peronosporalean oomycetes occurred without selection from ethaboxam yet confers insensitivity. We propose several evolutionary hypotheses for the repeated evolution of the C239S mutation.

Interaction of Brassicaceae Seed Meal Soil Amendment and Apple Rootstock Genotype on Microbiome Structure and Replant Disease Suppression.

Preplant soil application of a Brassica juncea-Sinapis alba seed meal formulation (SM) at a rate of 6.6 t ha-1 alters composition of the orchard soil microbiome in a manner that yields sustainable long-term suppression of soilborne pathogens in apple production systems. However, the cost of SM amendment has hindered the adoption of this tactic to manage apple replant disease in commercial orchards. Greenhouse trials were conducted to assess the effect of reduced SM application rates in concert with apple rootstock genotype on structure of the rhizosphere microbiome and associated disease control outcomes. At all application rates assessed, SM treatment increased tree growth and reduced disease development relative to the control. In general, total tree biomass and leader shoot length were similar in soils treated with SM at 4.4 or 6.6 t ha-1 regardless of rootstock genotype. Equivalent increase in tree biomass when cultivated in soil treated at the lowest and highest SM amendment rate was attained when used in conjunction with G.41 or G.210 apple rootstocks. Suppression of Pythium spp. or Pratylenchus penetrans root densities was similar at all SM application rates. When cultivated in nontreated replant orchard soil, Geneva rootstocks (G.41 and G.210) exhibited lower levels of Pythium spp. and P. penetrans root colonization relative to Malling rootstocks (M.9 and MM.106). For a given rootstock, structure of the rhizosphere microbiome was similar in soils treated with SM at 4.4 and 6.6 t ha-1. G.41 and G.210 rootstocks but not M.9 or MM.106 cultivated in soil treated with SM at 2.2 t ha-1 possessed a rhizosphere bacterial community structure that differed significantly from the control. Findings indicate that effective control of apple replant disease may be attained at lower SM amendment rates than employed previously, with lower effective rates possible when integrated with tolerant rootstock genotypes such as G.41 or G.210.

Bioactivity and action mechanism of green propolis against Pythium aphanidermatum.

We have established how natural compounds from green propolis collected by the species Apis mellifera act against the growth of Pythium aphanidermatum. On the basis of mass spectrometry (Q-ToF MS), we determined that Artepillin C, the major constituent of green propolis, underlies the effect and displays activity against P. aphanidermatum at a minimal inhibitory concentration of 750 µg.mL-1. Biophysical studies based on model membranes showed that this inhibitory effect may be linked with a membrane-related phenomenon: Artepillin C increases the permeability of membranes with relatively high fluidity in their lateral structure, a feature that is in line with the lipid composition reported for the cytoplasmic membrane of P. aphanidermatum. Therefore, the present study supports the use of the effective and inexpensive green propolis to control the impact of the dangerous phytopathogen P. aphanidermatum on agriculture.