High-Throughput Bioassay for Detection of Latent Fungi in Postharvest Produce.

Enormous fresh agricultural produce is wasted annually due to rots caused by pathogenic microorganisms. Most pathogenic fungi attack the harvested produce by penetrating the fruit at the field and remaining quiescent or latent until the fruit ripens or senescence. In this work, a recently developed simple, cost-effective, and high-throughput 96-well plate-based assay was applied to determine the presence of pathogenic fungi in their latent stage. The surface strands immobilized on the 96-well plate, only with the presence of the complementary RNA marker (enoyl-CoA hydratase (ECH)) of the latent fungal-pathogen Colletotrichum gloeosporioides will create a complex with the target and reporter (labeled with the horseradish peroxidase (HRP) enzyme) strands for positive signal generation. The developed assay demonstrated 3.1-fold higher specificity for the latent marker (ECH) of C. gloeosporioides compared to latent markers of other pathogenic fungi. A 2 nM detection limit of target strands was demonstrated, showing a high plate sensitivity, and was further validated with biological samples extracted from latent infection in tomato fruit. The developed assay provides a new economical tool for detecting the presence of latent RNA markers of pathogenic fungi in agricultural produce, ultimately improving postharvest decision-making and reducing postharvest losses.

Chitosan-based nanopesticides enhanced anti-fungal activity against strawberry anthracnose as "sugar-coated bombs".

A chitosan-based nanoparticle was prepared using chitosan (CS) and O-carboxymethyl chitosan (O-CMCS). Our study revealed that chitosan/O-carboxymethyl chitosan/tebuconazole nanoparticles (CS/O-CMCS/TBA NPs) exhibited superior antifungal activity, foliar adhesion, and microbial target adhesion performance compared to commercial suspension concentrate (SC). The antifungal activity of CS/O-CMCS/TBA NPs against C. gloeosporioides, with a 3.13-fold increase in efficacy over TBA (SC). We also found that low concentrations of CS/O-CMCS NPs promoted the growth of C. gloeosporioides and enhanced the fungal catabolism of chitosan. Overall, the CS/O-CMCS/TBA NPs were found to possess the remarkable capability to selectively aggregate around pathogenic microorganisms and CS/O-CMCS NPs can enhance the fungal catabolism of chitosan. CS/O-CMCS/TBA NPs, as a "sugar-coated bomb", was a promising asset for effective plant disease management and pesticide utilization through the affinity of chitosan-based nanoparticles and C. gloeosporioides, enabling targeted delivery and targeted release of their encapsulated active ingredient, which was important for the development and application of biocompatible chitosan-based nanopesticides.

Early on-site detection of strawberry anthracnose using portable Raman spectroscopy.

We developed a method for the early on-site detection of strawberry anthracnose using a portable Raman system with multivariate statistical analysis algorithms. By using molecular markers based on Raman spectra, the proposed method can detect anthracnose in strawberry stems 3 days after exposure to Colletotrichum gloeosporioides. A fiber-optic probe was applied for the portable Raman system, and the acquisition time was 10 s. We found that the molecular markers were closely related to the following subjects: i) an increase in amide III and fatty acids of C. gloeosporioides invading strawberry stems (Raman bands at 1180-1310 cm-1) and ii) a decrease in metabolites in strawberry plants, such as phenolic compounds and terpenoids (Raman bands at 760, 800, and 1523 cm-1). We also found that the increased fluorescence background caused by various chromophores within the invading C. gloeosporioides could serve as a marker. A two-dimensional cluster plot obtained by principal component analysis (PCA) showed that the three groups (control, fungal infection, and pathogen) were distinguishable. The linear discriminant analysis (LDA)-based prediction algorithm could identify C. gloeosporioides infection with a posterior probability of over 40%, even when no symptoms were visible on the inoculated strawberry plants.

Genomic Resources of Four Colletotrichum Species (C. fioriniae, C. chrysophilum, C. noveboracense, and C. nupharicola) Threatening Commercial Apple Production in the Eastern United States.

The genus Colletotrichum includes nine major clades with 252 species and 15 major phylogenetic lineages, also known as species complexes. Colletotrichum spp. are one of the top fungal plant pathogens causing anthracnose and pre- and postharvest fruit rots worldwide. Apple orchards are imperiled by devastating losses from apple bitter rot, ranging from 24 to 98%, which is a serious disease caused by several Colletotrichum species. Bitter rot is also a major postharvest rot disease, with C. fioriniae causing from 2 to 14% of unmarketable fruit in commercial apple storages. Dominant species causing apple bitter rot in the Mid-Atlantic United States are C. fioriniae from the Colletotrichum acutatum species complex and C. chrysophilum and C. noveboracense from the C. gloeosporioides species complex (CGSC). C. fioriniae is the dominant species causing apple bitter rot in the Northeastern and Mid-Atlantic states. C. chrysophilum was first identified on banana and cashew but has been recently found as the second most dominant species causing apple bitter rot in the Mid-Atlantic. As the third most dominant pathogen, C. noveboracense MB 836581 was identified as a novel species in the CGSC, causing apple bitter rot in the Mid-Atlantic. C. nupharicola is a sister group to C. fructicola and C. noveboracense, also causing bitter rot on apple. We deliver the resources of 10 new genomes, including two isolates of C. fioriniae, three isolates of C. chrysophilum, three isolates of C. noveboracense, and two isolates of C. nupharicola collected from apple fruit, yellow waterlily, and Juglans nigra. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Bitter Rot of Apple in the Mid-Atlantic United States: Causal Species and Evaluation of the Impacts of Regional Weather Patterns and Cultivar Susceptibility.

Apple growers in the Mid-Atlantic region of the United States have been reporting an increase in losses to bitter rot of apple and are requesting up-to-date management recommendations. Management is complicated by variations in apple cultivar susceptibility, temperature, rainfall, and biology of the Colletotrichum spp. that cause bitter rot. Over 500 apple fruit with bitter rot were obtained from 38 orchards across the Mid-Atlantic and the causal species were identified as Colletotrichum fioriniae and C. nymphaeae of the C. acutatum species complex and C. chrysophilum, C. noveboracense, C. siamense, C. fructicola, C. henanense, and C. gloeosporioides sensu stricto of the C. gloeosporioides species complex, the latter two being first reports. Species with faster in vitro growth rates at higher temperatures were more abundant in warmer regions of the Mid-Atlantic, while those with slower growth rates at higher temperatures were more abundant in cooler regions. Regional bloom dates are earlier and weather data show a gradual warming trend that likely influenced but was not necessarily the main cause of the recent increase in bitter rot in the region. A grower survey of apple cultivar susceptibility showed high variation, with the increase in acres planted to the highly susceptible cultivar Honeycrisp broadly corresponding to the increase in reports of bitter rot. These results form a basis for future studies on the biology and ecology of the Colletotrichum spp. responsible, and suggest that integrated bitter rot management must begin with selection of less-susceptible apple cultivars.

Volatile organic compounds (VOCs) from Bacillus subtilis CF-3 reduce anthracnose and elicit active defense responses in harvested litchi fruits.

In this study, we investigated the effects of volatile organic compounds (VOCs) produced by Bacillus subtilis CF-3 on the growth and development of Colletotrichum gloeosporioides and evaluated the elicitation of active defense responses in harvested litchi fruits. In vitro experiments were conducted to explore the bacteriostatic effect of VOCs in inhibiting pathogenic fungi by means of plate enthalpy test, scanning electron microscopy, transmission electron microscopy, and gas chromatography-mass spectrometry (GC-MS). The results showed that 2,4-di-tert-butylphenol and CF-3 24-h fermentation broth (24hFB) can significantly inhibit the germination of fungal spores, disrupt hyphal and cell morphology, and decrease cell membrane fluidity and integrity, resulting in the changes of indexes. In addition, the bacteriostasis of VOCs in the defensive ability of litchi fruits to C. gloeosporioides was studied, and it was shown that 2,4-di-tert-butylphenol and CF-3 24hFB can inhibit the activity of the pathogenic enzymes (pectinase and cellulase) secreted by C. gloeosporioides to reduce the decomposition of plant tissues, activate antioxidant enzymes (peroxidase, polyphenol oxidase, catalase, and superoxide dismutase) in the fruit to eliminate excessive reactive oxygen species in fruits in order to reduce plant cell damage and activate disease resistance enzymes (phenylalanineammonialyase, chitinases, ß-1,3-glucanase) to enhance the resistance of litchi fruits to C. gloeosporioides and inhibit its growth. This study investigated the bacteriostasis of VOCs in inhibiting C. gloeosporioides and inducing the resistance of litchi fruits, providing a theoretical basis for future applications.

Activation of the phenylpropanoid biosynthesis pathway reveals a novel action mechanism of the elicitor effect of chitosan on avocado fruit epicarp.

Secondary metabolites play an important role in the avocado fruit defense system. Phenolic compounds are the main biosynthesized metabolites of this system response. Our objective in this investigation was to evaluate the induction of specific metabolic pathways using chitosan as an elicitor. Extracts obtained from avocado in intermediate and consumption maturity stages treated with chitosan exhibited an increase in antifungal activity, which caused inhibition of mycelial growth and a decrease in sporulation as well as spore germination of Colletotrichum gloeosporioides. Additionally, RNA from epicarp of the fruits treated and untreated with chitosan was obtained in order to evaluate the expression of genes related to phenylpropanoids and the antifungal compound 1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene biosynthesis. An increased in gene expression of genes that participates in the phenylpropanoids route was observed during the stage of physiological fruit maturity, others genes such as Flavonol synthase (Fls), increased only in samples obtained from fruit treated with chitosan at consumption maturity. Our results reveal a new molecular mechanism where chitosan induces a specific accumulation of phenylpropanoids and antifungal diene; this partially explains avocado's resistance against fungal pathogens. Finally, we discuss the molecular connections between chitosan induction and gene expression to explain the biological events that orchestrate the resistance pathways in fruits.

The expression of the genes involved in redox metabolism and hydrogen peroxide balance is associated with the resistance of cowpea [Vigna unguiculata (L.) Walp.] to the hemibiotrophic fungus Colletotrichum gloeosporioides.

Correlations between the transcriptional responses of genes that encode superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxiredoxin (Prx) enzymes and Colletotrichum gloeosporioides development in cowpea leaves were assessed. Each of these genes is involved in the redox metabolism and hydrogen peroxide balance. Although electron microscopy revealed that conidia adhered to and germinated on the leaf cuticle, the inoculated cowpea leaves did not show any characteristic anthracnose symptoms. The adhered and germinated conidia showed irregular surfaces and did not develop further. This was apparently due to increased leaf H2O2 levels in response to inoculation with C. gloeosporioides. During the early stages post inoculation, cowpea leaves elevated the H2O2 content and modulated the defense gene expression, as well as associated pathways. During the later stages, the increased expression of the CuZnSODI and CuZnSODII genes suggested an active superoxide dismutation to further elevate H2O2 levels, which indicated that higher H2O2 content may function as a toxic agent that kills the fungus. The second increase in H2O2 production above the threshold level was correlated with the expression of the APXI, CATI, CATII, PrxIIBCD, and PrxIIE genes, which resulted in a coordinated pattern to establish an appropriate balance between H2O2 generation and scavenging. Therefore, appropriate H2O2 content in cowpea leaves inhibited C. gloeosporioides development and maintained intracellular redox homeostasis to avoid uncontrolled programmed cell death and necrosis in cowpea leaves.

Transcriptomic Analysis of Avocado Hass (Persea americana Mill) in the Interaction System Fruit-Chitosan-Colletotrichum.

Avocado (Persea americana) is one of the most important crops in Mexico as it is the main producer, consumer, and exporter of avocado fruit in the world. However, successful avocado commercialization is often reduced by large postharvest losses due to Colletotrichum sp., the causal agent of anthracnose. Chitosan is known to have a direct antifungal effect and acts also as an elicitor capable of stimulating a defense response in plants. However, there is little information regarding the genes that are either activated or repressed in fruits treated with chitosan. The aim of this study was to identify by RNA-seq the genes differentially regulated by the action of low molecular weight chitosan in the avocado-chitosan-Colletotrichum interaction system. The samples for RNA-seq were obtained from fruits treated with chitosan, fruits inoculated with Colletotrichum and fruits both treated with chitosan and inoculated with the fungus. Non-treated and non-inoculated fruits were also analyzed. Expression profiles showed that in short times, the fruit-chitosan system presented a greater number of differentially expressed genes, compared to the fruit-pathogen system. Gene Ontology analysis of differentially expressed genes showed a large number of metabolic processes regulated by chitosan, including those preventing the spread of Colletotrichum. It was also found that there is a high correlation between the expression of genes in silico and qPCR of several genes involved in different metabolic pathways.

Antifungal activity against colletotrichum acutatum and colletotrichum gloeosporioides of the major constituents from wood sawdust of platymiscium gracile benth / Actividad antifúngica contra colletotrichum acutatum y colletotrichum gloeosporioides de los constituyentes mayoritarios del aserrín de madera de platymiscium gracile benth

The tree tomato (Solanum betaceum Cav., Solanaceae) anthracnose, caused by the fungi Colletotrichum acutatum and Colletotrichum gloeosporioides, is the most important disease of this crop in Colombia for its wide distribution and the losses it causes. In the present work, the in vitro antifungal activity of the soluble fractions in n-hexane, dichloromethane, and ethyl acetate, and their major constituents from the sawdust of timber specie Platymiscium gracile Benth. (Fabaceae) against both fungi was evaluated. The n-hexane-soluble fraction exhibited the greatest inhibitory effect. The metabolites homopterocarpin (a pterocarpan, 0.39 percent dry weight), calycosin (an isoflavone, 2.01 percent) and scoparone (a coumarin, 1.48 percent) were isolated for the first time from wood sawdust of P. gracile. The structure of these compounds was determined by 1H and 13C NMR analyses. The three compounds tested showed significant antifungal activity.

La antracnosis del tomate de árbol (Solanum betaceum Cav., Solanaceae), ocasionada por los hongos Colletotrichum acutatum y Colletotrichum gloeosporioides, es la enfermedad más importante de este cultivo en Colombia por su amplia distribución y las pérdidas que ocasiona. En el presente trabajo se evaluó la actividad antifúngica in vitro de las fracciones solubles en n-hexano, diclorometano y acetato de etilo, y sus componentes mayoritarios, del aserrín de la especie maderable Platymiscium gracile Benth. (Fabaceae), contra ambos hongos. La fracción en n-hexano exhibió el mayor efecto inhibitorio. Los metabolitos homopterocarpina (un pterocarpano; 0.39 por ciento del peso seco de aserrín), calicosin (una isoflavona; 2.01 por ciento) y escoparona (una cumarina; 1.48 por ciento) se aislaron por primera vez desde el aserrín de madera de P. gracile empleando técnicas cromatográficas. La estructura de los compuestos se determinó por análisis de RMN de 1H y 13C. Los tres metabolitos mostraron una actividad antifúngica significativa contra ambos hongos.

Experimental protocol for the recovery and evaluation of bioactive compounds of tarbush against postharvest fruit fungi.

The aim of this study was to recover and evaluate in vitro the antifungal activity of bioactive compounds of tarbush Flourensia cernua against fruit postharvest fungi and their antioxidant capacity. A yield of 15% of bioactive compounds of tarbush was obtained by infusion method and heating using water as solvent. A concentration of 4000 mg/L showed a higher antioxidant activity against the ABTS radical (3.21 µMol/g) in comparison with the DPPH radical (7.62 µMol/g); however the DPPH radical showed a better correlation with the content of tannins. The BCT showed values of IC50 between 1519 and 3310 mg/L against Rhizopus stolonifer, Botrytis cinerea, Fusarium oxysporum and Colletotrichum gloeosporioides. Antifungal activity is attributable mainly to gallic acid and flavonoids identified by infrared and HPLC analysis. In this study, the BCT have shown to be a possible natural alternative of antioxidant and antifungal compounds for use against postharvest fruit fungi.

Potentiality of Bacillus subtilis as biocontrol agent for management of anthracnose disease of chilli caused by Colletotrichum gloeosporioides OGC1.

A soil bacterium, Bacillus subtilis, isolated from the rhizosphere of Chilli, showed high antagonistic activity against Colletotrichum gloeosporioides OGC1. A clear inhibition zone of 0.5-1 cm was observed in dual plate assay. Microscopic observations showed a clear hyphal lysis and degradation of fungal cell wall. In dual liquid cultures, the B. subtilis strain inhibited the C. gloeosporioides up to 100 % in terms of dry weight. This strain also produced a clear halo region on chitin agar medium plates containing 0.5 % colloidal chitin, indicating that it excretes chitinase. The strain also produced other mycolytic enzymes-glucanase and cellulase, demonstrated by a clear zone of hydrolysis of yeast cell wall glucan (YCW 0.1 % v/v) and carboxymethylcellulose (CMC 0.1 % v/v). In liquid cultures, the strain showed appreciable levels of chitinase, glucanase and cellulase activities and hydrolytic activity with C. gloeosporioides OGC1 mycelia as the substrate. The role of the B. subtilis strain in suppressing the fungal growth in vitro was studied in comparison with a UV mutant of that strain, which lacked both antagonistic and hydrolytic activity. The mycolytic enzyme mediated antagonism of B. subtilis was further demonstrated by heat inactivation (70-100 °C), treatment with trypsin and TCA of the crude enzyme extract which lacked antifungal property also. Treatment of the chilli seeds with Bacillus sp. culture showed 100 % germination index similar to the untreated seeds. The treatment of the seed with co-inoculation of the pathogen with Bacillus sp. culture showed 65 % reduction in disease incidence by the treatment as compared to the seed treated with pathogen alone (77.5 %).