Diversity and Toxigenicity of Fungi that Cause Pineapple Fruitlet Core Rot.
ABSTRACT: The identity of the fungi responsible for fruitlet core rot (FCR) disease in pineapple has been the subject of investigation for some time. This study describes the diversity and toxigenic potential of fungal species causing FCR in La Reunion, an island in the Indian Ocean. One-hundred-and-fifty fungal isolates were obtained from infected and healthy fruitlets on Reunion Island and exclusively correspond to two genera of fungi: Fusarium and Talaromyces. The genus Fusarium made up 79% of the isolates, including 108 F. ananatum, 10 F. oxysporum, and one F. proliferatum. The genus Talaromyces accounted for 21% of the isolated fungi, which were all Talaromyces stollii. As the isolated fungal strains are potentially mycotoxigenic, identification and quantification of mycotoxins were carried out on naturally or artificially infected diseased fruits and under in vitro cultures of potential toxigenic isolates. Fumonisins B1 and B2 (FB1-FB2) and beauvericin (BEA) were found in infected fruitlets of pineapple and in the culture media of Fusarium species. Regarding the induction of mycotoxin in vitro, F.proliferatum produced 182 mg kg⁻1 of FB1 and F. oxysporum produced 192 mg kg⁻1 of BEA. These results provide a better understanding of the causal agents of FCR and their potential risk to pineapple consumers.
Project description:Fruitlet Core Rot (FCR) is a fungal disease that negatively impacts the quality of pineapple, in particular the 'Queen Victoria' cultivar. The main FCR causal agent has been identified as <i>Fusarium</i><i>ananatum</i>. This study focused on the correlation between FCR disease occurrence, fungal diversity, and environmental factors. FCR incidence and fungal species repartition patterns were spatially contextualized with specific surrounding parameters of the experimental plots. The mycobiome composition of healthy and diseased fruitlets was compared in order to search for potential fungal markers. A total of 240 pineapple fruits were sampled, and 344 fungal isolates were identified as belonging to 49 species among 17 genera. FCR symptom distribution revealed a significant gradient that correlated to that of the most abundant fungal species. The association of wind direction and the position of proximal cultivated crops sharing pathogens constituted an elevated risk of FCR incidence. Five highly represented species were assayed by Koch's postulates, and their pathogenicity was confirmed. These novel pathogens belonging to <i>Fusarium</i><i>fujikuroi</i> and <i>Talaromyces</i><i>purpureogenus</i> species complexes were identified, unravelling the complexity of the FCR pathosystem and the difficulty of apprehending the pathogenesis over the last several decades. This study revealed that FCR is an airborne disease characterized by a multi-partite pathosystem.
Project description:Pineapple (Ananas comosus var. comosus) is an important perennial crop in tropical and subtropical areas. It may be infected by various Fusarium species, contaminating the plant material with mycotoxins. The aim of this study was to evaluate Fusarium species variability among the genotypes isolated from pineapple fruits displaying fungal infection symptoms and to evaluate their mycotoxigenic abilities. Forty-four isolates of ten Fusarium species were obtained from pineapple fruit samples: F. ananatum, F. concentricum, F. fujikuroi, F. guttiforme, F. incarnatum, F. oxysporum, F. polyphialidicum, F. proliferatum, F. temperatum and F. verticillioides. Fumonisins B1-B3, beauvericin (BEA) and moniliformin (MON) contents were quantified by high-performance liquid chromatography (HPLC) in pineapple fruit tissue. Fumonisins are likely the most dangerous metabolites present in fruit samples (the maximum FB1 content was 250 μg g(-1) in pineapple skin and 20 μg ml(-1) in juice fraction). In both fractions, BEA and MON were of minor significance. FUM1 and FUM8 genes were identified in F. fujikuroi, F. proliferatum, F. temperatum and F. verticillioides. Cyclic peptide synthase gene (esyn1 homologue) from the BEA biosynthetic pathway was identified in 40 isolates of eight species. Based on the gene-specific polymerase chain reaction (PCR) assays, none of the isolates tested were found to be able to produce trichothecenes or zearalenone.
Project description:<i>Fusarium</i> species are among the most important fungal pathogens of maize, where they cause severe reduction of yield and accumulation of a wide range of harmful mycotoxins in the kernels. In order to identify the <i>Fusarium</i> species and their mycotoxin profiles associated to maize ear rot and kernel contamination in Iran, a wide sampling was carried out from field in ten major maize-producing provinces in Iran, during 2015 and 2016. From 182 samples of maize kernels, 551 strains were isolated and identified as belonging to <i>Fusarium</i> genus. Among the 234 representative strains identified at species level by translation elongation factor (<i>EF-1α</i>) sequences, the main <i>Fusarium</i> species were <i>F. verticillioides</i> and <i>F. proliferatum</i>, together representing 90% of the Iranian <i>Fusarium</i> population, and, to a lesser extent, <i>F. incarnatum equiseti</i> species complex (FIESC)<i>, F. thapsinum</i> and <i>F. redolens</i>. Fumonisin (FBs) production by <i>F. verticillioides</i> and <i>F. proliferatum</i> representative strains was analysed, showing that all strains produced FB<sub>1</sub>. None of <i>F. verticillioides</i> strains produced FB<sub>2</sub> nor FB<sub>3</sub>, while both FB<sub>2</sub> and FB<sub>3</sub> were produced only by <i>F. proliferatum</i>. Total mean of FBs production by <i>F. verticillioides</i> was higher than <i>F. proliferatum</i>. The occurrence of different <i>Fusarium</i> species on Iranian maize is reason of great concern because of the toxigenic risk associated to these species. Moreover, the diversity of the species identified increases the toxigenic risk associated to <i>Fusarium</i> contaminated maize kernels, because of the high possibility that a multi-toxin contamination can occur with harmful consequences on human and animal health.
Project description:In 2017-2018, extensive symptoms of sudden decline and fruit rot were observed on date palms in southern Tunisia. Samples of diseased plants were randomly collected in six localities. Based on morphological identification, <i>Fusarium</i> was the most frequent fungal genus detected. A sequencing of translation elongation factor, calmodulin, and second largest subunit of RNA polymerase II genes was used to identify 63 representative <i>Fusarium</i> strains at species level and investigate their phylogenetic relationships. The main species detected was <i>Fusarium</i> <i>proliferatum</i>, and at a much lesser extent, <i>Fusarium</i> <i>brachygibbosum</i>, <i>Fusarium</i> <i>caatingaense</i>, <i>Fusarium</i> <i>clavum</i>, <i>Fusarium</i> <i>incarnatum</i><i>,</i> and <i>Fusarium</i> <i>solani</i>. Pathogenicity on the <i>Deglet</i> <i>Nour</i> variety plantlets and the capability to produce mycotoxins were also assessed. All <i>Fusarium</i> species were pathogenic complying Koch's postulates. <i>Fusarium</i> <i>proliferatum</i> strains produced mainly fumonisins (FBs), beauvericin (BEA), and, to a lesser extent, enniatins (ENNs) and moniliformin (MON). All <i>F.</i> <i>brachygibbosum</i> strains produced low levels of BEA, diacetoxyscirpenol, and neosolaniol; two strains produced also T-2 toxin, and a single strain produced HT-2 toxin. <i>Fusarium</i> <i>caatingaense</i>, <i>F.</i> <i>clavum</i>, <i>F.</i> <i>incarnatum</i> produced only BEA. <i>Fusarium</i> <i>solani</i> strains produced MON, BEA, and ENNs. This work reports for the first time a comprehensive multidisciplinary study of <i>Fusarium</i> species on date palms, concerning both phytopathological and food safety issues.
Project description:Fungi from the Hypocreales order synthesize a range of toxic non-ribosomal cyclic peptides with antimicrobial, insecticidal and cytotoxic activities. Entomopathogenic Beauveria, Isaria and Cordyceps as well as phytopathogenic Fusarium spp. are known producers of beauvericins (BEAs), beauvenniatins (BEAEs) or enniatins (ENNs). The compounds are synthesized by beauvericin/enniatin synthase (BEAS/ESYN1), which shows significant sequence divergence among Hypocreales members. We investigated ENN, BEA and BEAE production among entomopathogenic (Beauveria, Cordyceps, Isaria) and phytopathogenic (Fusarium) fungi; BEA and ENNs were quantified using an LC-MS/MS method. Phylogenetic analysis of partial sequences of putative BEAS/ESYN1 amplicons was also made. Nineteen fungal strains were identified based on sequence analysis of amplified ITS and tef-1? regions. BEA was produced by all investigated fungi, with F. proliferatum and F. concentricum being the most efficient producers. ENNs were synthesized mostly by F. acuminatum, F. avenaceum and C. confragosa. The phylogeny reconstruction suggests that ancestral BEA biosynthesis independently diverged into biosynthesis of other compounds. The divergent positioning of three Fusarium isolates raises the possibility of parallel acquisition of cyclic depsipeptide synthases in ancient complexes within Fusarium genus. Different fungi have independently evolved NRPS genes involved in depsipeptide biosynthesis, with functional adaptation towards biosynthesis of overlapping yet diversified metabolite profiles.
Project description:Beauvericin (BEA) is a cyclodepsipeptide mycotoxin, showing insecticidal, antibiotic and antimicrobial activities, as well as inducing apoptosis of cancer cell lines. BEA can be produced by multiple fungal species, including saprotrophs, plant, insect and human pathogens, particularly belonging to <i>Fusarium</i>, <i>Beauveria</i> and <i>Isaria</i> genera. The ability of <i>Trichoderma</i> species to produce BEA was until now uncertain. Biosynthesis of BEA is governed by a non-ribosomal peptide synthase (NRPS), known as beauvericin synthase (BEAS), which appears to present considerable divergence among different fungal species. In the present study we compared the production of beauvericin among <i>Fusarium</i> and <i>Trichoderma</i> strains using UPLC methods. <i>BEAS</i> fragments were sequenced and analyzed to examine the level of the gene's divergence between these two genera and confirm the presence of active <i>BEAS</i> copy in <i>Trichoderma</i>. Seventeen strains of twelve species were studied and phylogenetic analysis showed distinctive grouping of <i>Fusarium</i> and <i>Trichoderma</i> strains. The highest producers of beauvericin were <i>F. proliferatum</i> and <i>F. nygamai</i>. <i>Trichoderma</i> strains of three species (<i>T. atroviride</i>, <i>T. viride</i>, <i>T. koningiopsis</i>) were minor BEA producers. The study showed beauvericin production by <i>Fusarium</i> and <i>Trichoderma</i> species and high variance of the non-ribosomal peptide synthase gene among fungal species from the <i>Hypocreales</i> order.
Project description:<i>Fusarium</i> diseases, including corn root rot, sheath rot, stalk rot, and ear rot are frequently occurring in maize producing areas of China. <i>Fusarium</i> stalk rot and ear rot are the most serious diseases and often occur at the same time, but it is unclear whether there is a correlation between <i>Fusarium</i> composition and disease occurrence. This study was conducted to clarify the relationship between the two diseases. A total of 49 corn stalk rot samples were collected from 15 regions of eight provinces in China from 2016 to 2018. The pathogens were isolated and identified separately from stalks, ear stems, and kernels. The contents of the fumonisins (FB<sub>1</sub> and FB<sub>2</sub>) were detected in kernels. The results showed that the main <i>Fusarium</i> species were found in corn kernels, ear stems and stalks at the same time. The results showed that 1201 strains of <i>Fusarium verticillioides</i>, 668 strains of <i>Fusarium oxysporum</i>, 574 strains of <i>Fusarium graminearum</i> species complex (FGSC), 318 strains of <i>Fusarium equiseti</i>, 95 strains of <i>Fusarium proliferatum</i>, and 40 strains of <i>Fusarium subglutinans</i> were isolated from 1470 corn kernels, 245 ear stems, and 1225 stalks randomly selected from 49 samples. The contamination rate of fumonisins in the 49 samples was 57.1% with an average content of 1.9 ?g/g, of which four samples exhibited higher levels as set by the European Commission (4.0 ?g/g). These results provide a certain association between stalk rot and ear rot and lay a foundation to study the relationships among <i>Fusarium</i> maize diseases.
Project description:Fruitlet core rot is one of the major postharvest disease of pineapple (Ananas comosus var. comosus). In the past, control strategies were designed to eliminate symptoms without addressing their causes or mechanisms, thus achieving only moderate success. In this study, (i) we focused on the anatomy of the fruitlets in the resistant "MD-2" and susceptible "Queen" pineapple cultivars; (ii) we identified the key role of the carpel margin in the infection process; (iii) we identified the key role of the sinuous layer of thick-walled cells in the inhibition of Fusarium ananatum colonization; and (iv) we linked the anatomy of the fruitlets with the phenolic content of cell walls. The fruitlet anatomy of the two cultivars was studied using X-ray, fluorescence, and multiphoton microscopy. Sepals and bracts were not perfectly fused with each other, allowing the pathogen to penetrate the fruit even after flowering. In fact, the fungi were found in the blossom cups of both cultivars but only became pathogenic in the flesh of the "Queen" pineapple fruit under natural conditions. The outer layer of the "MD-2" cavity was continuous with thick cell walls composed of ferulic and coumaric acids. The cell walls of the "Queen" blossom cup were less lignified at the extremities, and the outer layer was interspersed with cracks. The carpel margins were fused broadly in the "MD-2" pineapple, in contrast to the "Queen" pineapple. This blemish allows the fungus to penetrate deeper into the susceptible cultivar. In pineapple fruitlets, the hyphae of F. ananatum mainly progressed directly between cell walls into the parenchyma but never reached the vascular region. A layer of thick-walled cells, in the case of the resistant cultivar, stopped the colonization, which were probably the infralocular septal nectaries. Anatomical and histochemical observations coupled with spectral analysis of the hypodermis suggested the role of lignin deposition in the resistance to F. ananatum. The major phenolics bound to the cell walls were coumaric and ferulic acids and were found in higher amounts in the resistant cultivar postinoculation. The combination of fruitlet anatomy and lignification plays a role in the mechanism of host resistance to fruitlet core rot.
Project description:Minimally-processed pineapple stored under refrigerated conditions is highly perishable. We aimed to characterize the evolution of physicochemical, sensory and microbiological quality during cold storage. Pineapple batches were sampled from several locations in Reunion Island and then minimally processed. In the processing step, the variability of firmness and counts of yeasts and molds were observed. Moreover, correlations between the sampling season and pH and b* color component, as well as between fungal population and b* parameter were observed. During storage, the visual aspect of pineapple cuts changed to brown and shiny, whereas olfactive descriptors shifted from fruity descriptors and fresh to fermented, alcoholic and milky. The values for pH, TA and TSS did not significantly vary according to storage time. A decrease in firmness and C* color parameter was observed. Yeast and mold counts were significantly higher after 7 days of storage. The diversity in yeasts and molds was mainly dependent on the considered batches observed from PCR-DGGE profiles. Fungal species were isolated from spoiled pineapple cuts. The implication of Penicilllium citrtrinum, Talaromyces amestolkiae, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, and Meyerozyma caribbica in the spoilage of minimally-processed pineapple cuts was further demonstrated.
Project description:The taxonomy of the genus <i>Fusarium</i> has been in a <i>flux</i> because of ambiguous circumscription of species-level identification based on morphotaxonomic criteria. In this study, multigene phylogeny was conducted to resolve the evolutionary relationships of 88 Indian <i>Fusarium</i> isolates based on the internal transcribed spacer region, 28S large subunit, translation elongation factor 1-alpha, RNA polymerase second largest subunit, beta-tubulin and calmodulin gene regions. <i>Fusarium</i> species are well known to produce metabolites such as beauvericin (BEA) and enniatins. These identified isolates were subjected to fermentation in <i>Fusarium</i>-defined media for BEA production and tested using TLC, HPLC and HRMS. Among 88 isolates studied, 50 were capable of producing BEA, which varied from 0.01 to 15.82 mg/g of biomass. <i>Fusarium tardicrescens</i> NFCCI 5201 showed maximum BEA production (15.82 mg/g of biomass). The extract of <i>F</i>. <i>tardicrescens</i> NFCCI 5201 showed promising antibacterial activity against <i>Staphylococcus aureus</i> MLS16 MTCC 2940 and <i>Micrococcus luteus</i> MTCC 2470 with MIC of 62.5 and 15.63 µg/mL, respectively. Similarly, the <i>F</i>. <i>tardicrescens</i> NFCCI 5201 extract in potato dextrose agar (40 µg/mL) exhibited antifungal activity in the food poison technique against plant pathogenic and other fungi, <i>Rhizoctonia solani</i> NFCCI 4327, <i>Sclerotium rolfsii</i> NFCCI 4263, <i>Geotrichum candidum</i> NFCCI 3744 and <i>Pythium</i> sp. NFCCI 3482, showing % inhibition of 84.31, 49.76, 38.22 and 35.13, respectively. The antibiotic effect was found to synergize when <i>Fusarium</i> extract and amphotericin B (20 µg/mL each in potato dextrose agar) were used in combination against <i>Rhizopus</i> sp. NFCCI 2108, <i>Sclerotium rolfsii</i> NFCCI 4263, <i>Bipolaris sorokiniana</i> NFCCI 4690 and <i>Absidia</i> sp. NFCCI 2716, showing % inhibition of 50.35, 79.37, 48.07 and 76.72, respectively. The extract also showed satisfactory dose-dependent DPPH radical scavenging activity with an IC<sub>50</sub> value of 0.675 mg/mL. This study reveals the correct identity of the Indian <i>Fusarium</i> isolates based on multigene phylogeny and also throws light on BEA production potential, suggesting their possible applicability in the medicine, agriculture and industry.