Penicillium digitatum infection mechanisms in citrus: What do we know so far?

Penicillium digitatum is the major source of postharvest decay in citrus fruits worldwide. This fungus shows a limited host range, being able to infect mainly mature fruit belonging to the Rutaceae family. This highly specific host interaction has attracted the interest of the scientific community. Researchers have investigated the chemical interactions and specialized virulence strategies that facilitate this fungus's fruit colonization, thereby leading to a successful citrus infection. There are several factors that mediate and affect the interaction between P. digitatum and its host citrus, including hydrogen peroxide modulation, secretion of organic acids and consequently pH control, and other strategies described here. The recently achieved sequencing of the complete P. digitatum genome opened up new possibilities for exploration of the virulence factors related to the host-pathogen interaction. Through such techniques as RNAseq, RT-PCR and targeted gene knockout mediated by Agrobacterium tumefaciens, important genes involved in the fungal infection process in citrus have been reported, helping to elucidate the molecular mechanisms, metabolites and genetic components that are involved in the pathogenicity of P. digitatum. Understanding the infection process and fungal strategies represents an important step in developing ways to protect citrus from P. digitatum infection, possibly leading to more productive citriculture.     

Biodiversity of Convolvulaceous species that contain ergot alkaloids,indole diterpene alkaloids,and swainsonine

Convolvulaceous species have been reported to contain several bioactive principles thought to be toxic to livestock including the calystegines, swainsonine, ergot alkaloids, and indole diterpene alkaloids. Swainsonine, ergot alkaloids, and indole diterpene alkaloids are produced by seed transmitted fungal symbionts associated with their respective plant host, while the calystegines are produced by the plant. To date, Ipomoea asarifolia and Ipomoea muelleri represent the only Ipomoea species and members of the Convolvulaceae known to contain indole diterpene alkaloids, however several other Convolvulaceous species are reported to contain ergot alkaloids. To further explore the biodiversity of species that may contain indole diterpenes, we analyzed several Convolvulaceous species (n = 30) for indole diterpene alkaloids, representing four genera, Argyreia, Ipomoea, Stictocardia, and Turbina, that had been previously reported to contain ergot alkaloids. These species were also verified to contain ergot alkaloids and subsequently analyzed for swainsonine. Ergot alkaloids were detected in 18 species representing all four genera screened, indole diterpenes were detected in two Argyreia species and eight Ipomoea species of the 18 that contained ergot alkaloids, and swainsonine was detected in two Ipomoea species. The data suggest a strong association exists between the relationship of the Periglandula species associated with each host and the occurrence of the ergot alkaloids and/or the indole diterpenes reported here. Likewise there appears to be an association between the occurrence of the respective bioactive principle and the genetic relatedness of the respective host plant species.     

The completed genome sequence of the pathogenic ascomycete fungus Penicillium digitatum

P. digitatum, the causative agent of green mold, is one of the most destructive pathogens in the citrus industry. To facilitate basal researches on this important plant pathogen, here we report a finished genome sequence for P. digitatum strain PDW03 using a combination of Illumina, PacBio, and Hi-C sequencing technologies. The assembly comprised 6 chromosomes from telomere to telomere and encodes approximately 9000 proteins. Genomic re-analyses identified 302 Carbohydrate-active enzymes, 420 secreted proteins, and 39 secondary metabolite (SM) gene clusters. Furthermore, we found 10 fragmentary SM clusters in the P. digitatum PDW03 genome. Pangenome analysis based on 5 P. digitatum genomes available showed that conserved orthogroups account for ~68% of the species pangenome. Taken together, this fully completed P. digitatum genome will provide an optimum resource for further researches to investigate the driving forces of fungal host switch and effectors functioning in plant-pathogen interaction.     

Bio-guided search of active indole alkaloids from Tabernaemontana catharinensis: Antitumour activity,toxicity in silico and molecular modelling studies

Active plant metabolites have been used as prototype drugs. In this context, Tabernaemontana catharinensis (Apocynaceae) has been highlighted because of the presence of active indole alkaloids. Thus, this study aims the bio-guided search of T. catharinensis cytotoxic alkaloids. The chemical composition was identified by high-resolution mass spectrometry, and fractionation was performed by open column and preparative thin-layer chromatography, from plant stems. The enriched fractions were tested in vitro in tumour cells A375 (melanoma cell line) and A549 (adenocarcinomic human alveolar basal epithelial cells), and non-tumour Vero cells (African green monkey kidney epithelial cells). The alkaloids identified as active were submitted to in silico toxicity prediction by ADME-Tox and OSIRIS programs and, also, to molecular docking, using topoisomerase I (PDB ID: 1SC7) by iGEMDOCK. As a result, six sub-fractions were obtained, which were identified as containing 16-epi-affinine, 12-methoxy-n-methyl-voachalotine, affinisine, voachalotine, coronaridine hydroxyindoline and ibogamine, respectively. The affinisine-containing sub-fraction showed selective toxicity against A375, with an IC₅₀ of 11.73 µg mL⁻¹, and no cytotoxicity against normal cells (Vero). From the in silico toxicity test results, all indole alkaloid compounds had a low toxicity risk. The molecular docking data provided structural models and binding affinities of the plant’s indole alkaloids and topoisomerase I. In summary, this bio-guided search revealed that the indole alkaloids from T. catharinensis display selective cytotoxicity in A375 tumour cells and toxicity in silico. Particularly, affinisine might be a chemotherapeutic for A375 melanoma cells.     

“Self” and “Non-Self” in the Control of Phytoalexin Biosynthesis: Plant Phospholipases A2 with Alkaloid-Specific Molecular Fingerprints

The overproduction of specialized metabolites requires plants to manage the inherent burdens, including the risk of self-intoxication. We present a control mechanism that stops the expression of phytoalexin biosynthetic enzymes by blocking the antecedent signal transduction cascade. Cultured cells of Eschscholzia californica (Papaveraceae) and Catharanthus roseus (Apocynaceae) overproduce benzophenanthridine alkaloids and monoterpenoid indole alkaloids, respectively, in response to microbial elicitors. In both plants, an elicitor-responsive phospholipase A2 (PLA2) at the plasma membrane generates signal molecules that initiate the induction of biosynthetic enzymes. The final alkaloids produced in the respective plant inhibit the respective PLA, a negative feedback that prevents continuous overexpression. The selective inhibition by alkaloids from the class produced in the “self” plant could be transferred to leaves of Nicotiana benthamiana via recombinant expression of PLA2. The 3D homology model of each PLA2 displays a binding pocket that specifically accommodates alkaloids of the class produced by the same plant, but not of the other class; for example, C. roseus PLA2 only accommodates C. roseus alkaloids. The interaction energies of docked alkaloids correlate with their selective inhibition of PLA2 activity. The existence in two evolutionary distant plants of phospholipases A2 that discriminate “self-made” from “foreign” alkaloids reveals molecular fingerprints left in signal enzymes during the evolution of species-specific, cytotoxic phytoalexins.     

The Spatial Distribution of Alkaloids in Psychotria prunifolia (Kunth) Steyerm and Palicourea coriacea (Cham.) K. Schum Leaves Analysed by Desorption Electrospray Ionisation Mass Spectrometry Imaging

Introduction

Species of the genera Psychotria and Palicourea are sources of indole alkaloids, however, the distribution of alkaloids within the plants is not known. Analysing the spatial distribution using desorption electrospray ionisation mass spectrometry imaging (DESI‐MSI) has become attractive due to its simplicity and high selectivity compared to traditional histochemical techniques.

Objectives

To apply DESI‐MSI to visualise the alkaloid distribution on the leaf surface of Psychotria prunifolia and Palicourea coriacea and to compare the distributions with HPLC–MS and histochemical analyses.

Methodology

Based upon previous structure elucidation studies, four alkaloids targeted in this study were identified using high resolution mass spectrometry by direct infusion of plant extracts, and their distributions were imaged by DESI‐MSI via tissue imprints on a porous Teflon surface. Relative quantitation of the four alkaloids was obtained by HPLC–MS/MS analysis performed using multiple‐reaction monitoring (MRM) mode on a triple quadrupole mass spectrometer.

Results

Alkaloids showed distinct distributions on the leaf surfaces. Prunifoleine was mainly present in the midrib, while 10‐hydroxyisodeppeaninol was concentrated close to the petiole; a uniform distribution of 10‐hydroxyantirhine was observed in the whole leaf of Psychotria prunifolia. The imprinted image from the Palicourea coriacea leaf also showed a homogeneous distribution of calycanthine throughout the leaf surface.

Conclusion

Different distributions were found for three alkaloids in Psychotria prunifolia, and the distributions found by MSI were in complete accordance with HPLC–MS analysis and histochemical results. The DESI‐MSI technique was therefore demonstrated to provide reliable information about the spatial distribution of metabolites in plants. Copyright © 2017 John Wiley & Sons, Ltd.     

Control of green mould of citrus by using Trichoderma harzianum,humic acid or garlic

Penicillium digitatum, an aggressive fungus causes post-harvest decay of mandarin sweet orange and Washington navel. In vitro Trichoderma harzianum or humic acid (HA) or powdered cloves of garlic caused inhibition of fungal growth of isolates P₁ and P₂. Under storage conditions, the fruit citrus is protected by using T. harzianum with standard volume 2.0?ml (9.6?×?10⁶?conidia/ml) and application 24?h before inoculation reduces disease incidence and disease severity after seven?days from inoculation with P. digitatum spore suspension (1.0?×?10⁶?spores/ml) compared to control. Spraying the fruit citrus by standard volume of 2.0?ml of either HA or powder cloves of garlic 1% on each fruit 24?h before inoculation reduces disease incidence and disease severity after seven?days from inoculation with P. digitatum (1.0?×?10⁶ spores/ml) compared to control. The lowest percentage of disease incidence and disease severity were associated with powder of cloves garlic and followed by HA and T. harzianum during two growing seasons compared with the untreated and control.     

Novel mutations in CYP51B from Penicillium digitatum involved in prochloraz resistance

Green mold caused by Penicillium digitatum is one of the most serious postharvest diseases of citrus fruit, and it is ubiquitous in all citrus growing regions in the world. Sterol 14α-demethylase (CYP51) is one of the key enzymes of sterol biosynthesis in the biological kingdom and a prime target of antifungal drugs. Mutations in CYP51s have been found to be correlated with resistance to azole fungicides in many fungal species. To investigate the mechanism of resistance to prochloraz (PRC) in P. digitatum, the PRC sensitivity was determined in vitro in this study to assess the sensitivity of 78 P. digitatum isolates collected in Hubei province. The results showed that 25 isolates were prochloraz-resistant (PRC-R), including six high-resistant (HR) strains, twelve medium-resistant (MR) and seven low-resistant (LR) strains. A sequence analysis showed no consistent point mutations of PdCYP51A in the PRC-R strains, but four substitutions of CYP51B were found, Q309H in LR strains, Y136H and Q309H in HR strains, and G459S and F506I in MR strains, which corresponded to the four sensitivity levels. Based on the sequence alignment analysis and homology modeling followed by the molecular docking of the PdCYP51B protein, the potential correlation between the mutations and PRC resistance is proposed.     

Juvenile hormone antagonistic activity of secondary metabolites from Streptomyces lactacystinicus and their insecticidal activity against Plutella xylostella

Due to their specificity to target insects and low toxicity to non-target organisms, insect growth regulators (IGRs) have been promising alternatives to neurotoxic insecticides. Actinobacteria produce a wide range of secondary metabolites with insecticidal and insect growth regulatory activities. In this study, the culture media of 25 actinobacteria isolates showing high juvenile hormone antagonist (JHAN) activity were assessed for their insecticidal activity to identify novel IGR compounds toxic to Plutella xylostella. Among them, four isolates exhibited high insecticidal activity against 3rd instar larvae of P. xylostella. Two isolates of IMBL-1412 and IMBL-1823 showing relatively high insecticidal activities (greater than90% mortality) were identified as Streptomyces lactacystinicus based on colony color on various International Streptomyces Project (ISP) media and nucleotide sequences of the 16S rRNA gene. The ethyl acetate fractions of both isolates showed high JHAN and insecticidal activities against P. xylostella larvae at a concentration of 100 ppm when the culture media of these two isolates were extracted sequentially using hexane, ethyl acetate, and butyl alcohol. These results suggested that secondary metabolites of these actinobacterial isolates could be efficiently applied to develop novel IGR insecticides for the control of P. xylostella.     

Therapeutic potential of indole alkaloids in respiratory diseases: A comprehensive review

BackgroundIndole alkaloids are very promising for potential therapeutic purposes and appear to be particularly effective against respiratory diseases. Several experimental studies have been performed, both in vivo and in vitro, to evaluate the effectiveness of indole alkaloids for the management of respiratory disorders, including asthma, emphysema, tuberculosis, cancer, and pulmonary fibrosis.PurposeThe fundamental objective of this review was to summarize the in-depth therapeutic potential of indole alkaloids against various respiratory disorders.Study designIn addition to describing the therapeutic potential, this review also evaluates the toxicity of these alkaloids, which have been utilized for therapeutic benefits but have demonstrated toxic consequences. Some indole alkaloids, including scholaricine, 19-epischolaricine, vallesamine, and picrinine, which are derived from the plant Alstonia scholaris, have shown toxic effects in non-rodent models.MethodsThis review also discusses clinical studies exploring the therapeutic efficacy of indole alkaloids, which have confirmed the promising benefits observed in vivo and in vitro.ResultsThe indole alkaloidal compounds have shown efficacy in subjects with respiratory diseases.ConclusionThe available data established both preclinical and clinical studies confirm the potential of indole alkaloids to treat the respiratory disorders.     

Functional identification of the DHN melanin synthesis gene cluster and its role in UV-C tolerance in citrus postharvest pathogenic fungus Penicillium digitatum

The filamentous fungus Penicillium digitatum brings out great losses in citrus fruits by causing citrus green mold disease during the postharvest period. Previously, we obtained a T-DNA insertion mutant N2130 of P. digitatum, which produced albino conidia. To understand the role of green-grey conidial pigment in P. digitatum, we identified the insertion site and deeply explored the 1,8-dihydroxynaphtsalene (DHN)-melanin synthesis gene cluster in this phytopathogen. In this study, we deleted five genes in P. digitatum, PdPksP, PdAbr1, PdArp1, PdArp2, and PdAyg1, and the experiments were further performed on phenotype analyses, including pigmentation, UV-C tolerance, virulence, growth rate, conidiation, stress (osmotic-, oxidative-, cell wall disturbing-, and high temperature-) tolerance, fungicide resistance, and conidial hydrophobicity. The results showed that the five deletion mutants (ΔPdPksP, ΔPdAbr1, ΔPdArp1, ΔPdArp2 and ΔPdAyg1) produced albino, brownish, brown, reddish-brown, and Yellowish green conidia, respectively. In addition, the survival colony forming units (CFUs) of the deletion mutants, under the treatment of UV-C radiation (261.4 mJ/cm²), were 0.3- to 0.6-fold of those surviving in wild-type strain N1. Moreover, after 522.8 mJ/cm²-UV-C-irradiation on conidia, the deletion mutants showed a larger decrease in pathogenicity on Valencia Orange fruits compared with strain N1. However, there were no significant differences among other phenotypes tested in this study. Collectively, our research reported the DHN-melanin synthesis pathway in P. digitatum for the first time, and revealed that DHN-melanin is important for P. digitatum to tolerate UV-C irradiation.     

The Cytogenetic Map of the Poncirus trifoliata (L.) Raf.��A Nomenclature System for Chromosomes of All Citric Species

A cytogenetic map was established for the trifoliate orange, Poncirus trifoliata. Chromosome size, centromere position and CMA⁺ heterochromatin content were determined for each chromosome pair, together with the position of BAC clones previously described as chromosome-specific markers. Although P. trifoliata is a true biological species, heteromorphisms for the size of the heterochromatic bands in two chromosome pairs were observed. A minimum set of four BACs was proposed for chromosome identification in P. trifoliata, as well as a chromosome nomenclature that can be applied to all citrus species. The Ctv locus was assigned to chromosome 9 and the remaining linkage groups can now be integrated to this physical map. This standard nomenclature for trifoliate orange will be valuable for genome-sequencing projects in all citrus species.     

The infection of oranges by Trichoderma viride and mixed infection by Trichoderma viride and Penicillium digitatum

Trichoderma viride spores applied in water to apparently uninjured skin of oranges do not cause lesions. Adding orange juice, rind extract, citric acid or orange essential oil did not influence infection. Oranges became infected only when the stem-end cuts or wounds deeper than 6 mm into oil vesicles were inoculated. Sound oranges in contact with decayed oranges did not become infected. Diphenyl-impregnated wrappers reduced infection. A mixed inoculum of T. viride and Penicillium digitatum caused as fast rotting as P. digitatum, which caused faster rotting than T. viride alone. Lesions infected with P. digitatum could become infected by T. viride but those caused by T. viride did not become infected by P. digitatum. T. viride was antagonistic to P. digitatum in vivo and in vitro, possibly because it produces a heat-labile diffusible substance toxic to P. digitatum.     

Chemistry and biological activity of azoprenylated secondary metabolites

N-Prenyl secondary metabolites (isopentenylazo-, geranylazo-, farnesylazo- and their biosynthetic derivatives) represent a family of extremely rare natural products. Only in recent years have these alkaloids been recognized as interesting and valuable biologically active secondary metabolites. To date about 35 alkaloids have been isolated from plants mainly belonging to the Rutaceae family, and from fungi, bacteria, and/or obtained by chemical synthesis. These metabolites comprise anthranilic acid derivatives, diazepinones, and indole, and xanthine alkaloids. Many of the isolated prenylazo secondary metabolites and their semisynthetic derivatives are shown to exert valuable in vitro and in vivo anti-cancer, anti-inflammatory, anti-bacterial, anti-viral, and anti-fungal effects. The aim of this comprehensive review is to examine the different types of prenylazo natural products from a chemical, phytochemical and biological perspective.     

Dereplication of Palicourea sessilis ethanol extracts by UPLC-DAD-ESI-MS/MS discloses the presence of hydroxycinnamic acid amides and the absence of monoterpene indole alkaloids

Secondary metabolites characterization of ethanol extracts of Palicourea sessilis leaves and stems by UPLC-DAD-ESI-MS/MS led to putative identification of hydrolysable tannins in leaf extract (ESI negative mode) while hydroxycinnamic acid amides (HCA) such as N-p-coumaroylputrescine and N-feruloylagmatine were detected in both leaf and stems extracts in the ESI positive mode. Secondary metabolites quantification data showed a higher content of total phenolic in the leaf extract while the total alkaloids contents are statistically equivalent in both of the extracts. Furthermore, monoterpene indole alkaloids were not detected in both extracts. The presence of HCA is here firstly reported for a Palicourea species. This finding increases the classes of secondary metabolites occurring in this genus.     

Potential for Development of Tolerance by Penicillium digitatum and Penicillium italicum after Repeated Exposure to Potassium Sorbate

Two strains of Penicillium digitatum and one strain of Penicillium italicum were exposed to various levels of sorbic acid and potassium sorbate, and the MICs were determined. Selected strains of the molds were then repeatedly exposed to subinhibitory levels of the compounds to determine whether increased tolerance might develop. The MIC of sorbic acid (pH 4.75) to P. digitatum was between 0.02 and 0.025%. The MIC of sorbate (pH 5.5) to two strains of P. digitatum and P. italicum was found to be between 0.06 and 0.08%. Increasing levels of sorbate resulted in increasing growth suppression of the molds. Populations of P. digitatum were tested for increased tolerance to sorbic acid, and none was found. Individual molds that started from the same parent colony were examined for increased tolerance to potassium sorbate. Two P. digitatum strains developed no observable increased tolerance, but P. italicum developed a slight increase in tolerance to sorbate. When spores of P. italicum and P. digitatum were exposed to higher levels of sorbate for prolonged times, the fungicidal or fungistatic activity of the inhibitor was dependent upon pH, length of exposure time, level of sorbate, and the mold strain.     

Enantioselective inhibitory abilities of enantiomers of notoamides against RANKL-induced formation of multinuclear osteoclasts

The marine-derived Aspergillus protuberus MF297-2 and the terrestrial A. amoenus NRRL 35600 produce enantiomeric prenylated indole alkaloids. Investigation of biological activities of the natural and synthetic derivatives revealed that (?)-enantiomers of notoamides A and B, 6-epi-notoamide T, and stephacidin A inhibited receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)–induced osteoclastogenic differentiation of murine RAW264 cells more strongly than their respective (+)-enantiomers. Among them, (?)-6-epi-notoamide T was the most potent inhibitor with an IC₅₀ value of 1.7 μM.     

Effect of some essential oils on mycelial growth of <Emphasis Type="Italic">Penicillium digitatum</Emphasis> Sacc.

The antifungal action of four essential oils of Foeniculum vulgare (fennel), Thymus vulgaris (thyme), Eugenia caryophyllata (Clove) and Salvia officinalis (sage) was tested in vitro against Penicillium digitatum Sacc. Direct contact and vapour phase were used to test the antifungal activity of these essential oils against P. digitatum that is responsible for green mould rot of citrus fruits. The vapour phase and direct contact of clove and thyme essential oils exhibited the strongest toxicity and totally inhibited the mycelial growth of the test fungus. Thyme and clove essential oils completely inhibited P. digitatum growth either when added into the medium 600 μl l⁻¹ or by their volatiles with 24 μl per 8 cm diameter Petri dish. In in vitro mycelial growth assay showed fungistatic and fungicidal activity by clove and thyme essential oils. Sage and fennel oils did not show any inhibitory activity on this fungus. Scanning electron microscopy (SEM) was done to study the mode of action of clove oil in P. digitatum and it was observed that treatment with the oil leads to large alterations in hyphal morphology.