Essential oils inhibiting Alternaria alternata and Colletotrichum gloeosporioides: a review.

Essential oils (EOs) have been investigated for their effectiveness against fungal fruit pathogens. The present review article summarises the EOs that inhibit Alternaria alternata and Colletotrichum gloeosporioides in the pre- and post-harvest stages of fruits. Thirty-nine scientific papers focusing on the extraction conditions and the antifungal activity of EOs were selected. The retrieved studies came mainly from China and Brazil. Hydrodistillation has been identified as the most used extractive method. The yields and chemical profiles were variable among the species. The in vitro studies were larger than the in vivo studies. The application of EOs reduced the incidence of fungal diseases in tomatoes (Lycopersicon esculentum), papaya (Carica papaya) and mango (Mangifera indica). EOs resulted as a potential ecological alternative for treating fungal diseases in fruits requiring further investigation.

Antifungal activity of Serratia plymuthica against the phytopathogenic fungus Alternariatenuissima.

The antifungal activity of Serratia plymuthica CCGG2742, a bacterial strain isolated from grapes berries skin, against a phytopathogenic fungus isolated from blueberries was evaluated in vitro and in vivo. In order to characterize the wild fungal isolate, phylogenetic analysis using concatenated DNA sequences from the RPB2 and TEF1 genes and of the ITS region was performed, allowing the identification of the fungal isolate that was called Alternaria tenuissima CC17. Hyphae morphology, mycelium ultrastructure, conidia and reproductive structures were in agreement with the phylogenetic analysis. The antifungal activity of the S. plymuthica strain was dependent on the composition of the culture medium. The greatest inhibition of mycelial growth of A. tenuissima CC17 by S. plymuthica CCGG2742 was observed on YTS medium, which lacks of an easily assimilable carbon source. Fungal growth medium supplemented with 50 % of bacterial supernatant decreased the conidia germination of A. tenuissima CC17 up to 32 %. Preventive applications of S. plymuthica CCGG2742 to blueberries and tomato leaves at conidia:bacteria ratio of 1:100, protected in 77.8 ± 4.6 % and 98.2 ± 0.6 % to blueberries and tomato leaves from infection caused by A. tenuissima CC17, respectively. To the best of our knowledge, this is the first report on the antifungal activity of S. plymuthica against A. tenuissima, which could be used as a biological control agent of plant diseases caused by this fungal species. In addition, the results of this work could be a starting point to attribute the real importance of A. tenuissima as a pathogen of blueberries in Chile, which until now had been considered almost exclusively to A. alternata. Likewise, this research could be relevant to start developing highly effective strategies based on S. plymuthica CCGG2742 for the control of this important phytopathogenic fungus.

[Identification and antifungal activity of halophilic bacteria isolated from saline soils in Campeche, México]. / Identificación y actividad antifúngica de bacterias halófilas aisladas de suelos salinos en Campeche, México.

Phytopathogenic fungi Alternaria alternata and Colletotrichum gloeosporioides cause diseases in plant tissues as well as significant postharvest losses. The use of chemical fungicides for their control has negative effects on health and the environment. Secondary metabolites from halophilic bacteria are a promising alternative for new antifungal compounds. In the present study, halophilic bacteria were isolated and characterized from two sites with saline soils called branquizales in Campeche, Mexico. A total of 64 bacteria were isolated. Agrobacterium, Bacillus, Inquilinus, Gracilibacillus, Metabacillus, Neobacillus, Paenibacillus, Priestia, Staphylococcus, Streptomyces and Virgibacillus were among the identified genera. The antifungal potential of the culture supernatant (CS) of 39 halophilic bacteria was investigated against C. gloeosporioides and A. alternata. The bacteria showing the greatest inhibition of mycelial growth corresponded to Bacillus subtilis CPO 4292, Metabacillus sp. CPO 4266, Bacillus sp. CPO 4295 and Bacillus sp. CPO 4279. The CS of Bacillus sp. CPO 4279 exhibited the highest activity and its ethyl acetate extract (AcOEt) inhibited the germination of C. gloeosporioides, with IC50 values of 8,630µg/ml and IC90 of 10,720µg/ml. The organic partition of the AcOEt extract led to three fractions, with acetonitrile (FAcB9) showing the highest antifungal activity, with values exceeding 66%. Halophilic bacteria from 'blanquizales' soils of the genus Bacillus sp. produce metabolites with antifungal properties that inhibit the phytopathogenic fungus C. gloeosporioides.

Characterization of antifungal properties of avocado leaves and majagua flowers extracts and their potential application to control Alternaria alternata.

Plant extracts are used as an alternative to a wide range of foods against different types of fungal pathogens. In the present study, the extracts of avocado leaves (Persea americana) and majagua flowers (Talipariti elatum) were tested according to their antifungal activity against different fungi. The most promising extracts were those of majagua flowers that were applied lyophilized and in aqueous extract, being very effective against Alternaria alternata and reaching a 50 % in vitro reduction. Antifungal properties were also evaluated during infection of apples by A. alternata. A decrease in infection progression was confirmed with up to a 30 % reduction in disease incidence and a 20 % reduction in disease severity. Majagua extracts were also tested combined with edible pectin coatings, greatly increasing their effectiveness up 60 % reduction. Thus, extracts of majagua could provide a feasible alternative to control fungal pathogens during postharvest.

Chemical composition of five essential oils and their antioxidant and in vitro and in vivo antifungal activities against Alternaria alternata in tomato crop.

This study aimed to determine the chemical composition of essential oils from A. gratíssima, O. basilicum, S. microphylla, T. riparia, and T. vulgaris and their antioxidant and antifungal activities against Alternaria alternata strains. Gas chromatography-mass spectrometry revealed that the major components of the essential oils were terpenes. The essential oils of S. microphylla and T. riparia showed higher antioxidant activities than the others. The essential oils of S. microphylla and T. riparia inhibited the growth of the fungus at 3.10 and 6.05 µL/mL, respectively. The essential oil of T. riparia inhibited 93.9% of the growth of the fungus and showed the highest in vivo efficiency in severity reduction (76.2%). We conclude that the essential oil of T. riparia shows promising antifungal activity and is an environmentally safe alternative for controlling fungal diseases in vegetables.

Characterization of Alternaria alternata and Alternaria scrophulariae Brown Spot in Colombian quinoa (Chenopodium quinoa).

Alternaria is a saprophytic and opportunistic fungus with a worldwide distribution that can affect the quality of various agricultural products, such as fruits, cereals, and pseudocereals. This research was carried out to investigate the population of this genus associated with quinoa cultivation in plots located in the Boyacá department (Colombia), the country's third-largest quinoa-producing department. The present study found 17 Alternaria isolates, of which 13 were identified as A. alternata and 4 as A. scrophulariae (formerly A. conjuncta) employed molecular markers of internal transcribed spacer (ITS) region and translation elongation factor 1α (TEF-1α). In the pathogenicity test under greenhouse conditions, all the Alternaria isolates showed some degree of pathogenicity on Piartal quinoa cultivar plants although no significant differences were found in isolates. The severity indices ranged from 2 to 5, and the percentage of affected leaves per plant ranged between 15% and 40%. This fungus affected the foliar tissue of quinoa, resulting in chlorotic and necrotic spots, symptoms that can generate a reduction in the quality and productivity of crops. This is the first time that the pathogenicity of Alternaria spp. in the Piartal variety has been described and the first report of this genera in quinoa crops of Colombia.

In vitro and Quantitative and Structure Activity Relationship (QSAR) evaluation of the antifungal activity of terpenoid constituents of essential oils against Alternaria alternata and Fusarium oxysporum / Evaluación in vitro y QSAR (Quantitative and Structure-Activity Relationship) de la actividad antifúngica de terpenoides obtenidos de aceites esenciales frente a Alternaria alternata y Fusarium oxysporum

INTRODUCTION: Fungal genera Alternaria and Fusarium include human and plant pathogenic species. Several antifungals have been used for their control, but excessive use has contributed to resistance development in pathogens. An alternative to searching for and developing new antifungal agents is using essential oils and their main components, which have biological activities of interest in medicine and food production. OBJECTIVE: To evaluate in vitro and in silico the antifungal activities of terpenoids against Alternaria alternata and Fusarium oxysporum. MATERIALS AND METHODS: The minimum inhibitory concentration and minimum fungicidal concentration values of 27 constituents of essential oils used against Alternaria alternata and Fusarium oxysporum were evaluated in vitro. In addition, using genetic algorithms, quantitative models of the structure-activity relationship were used to identify the structural and physicochemical properties related to antifungal activity. RESULTS: The evaluated compounds proved to be effective antifungals. Thymol was the most active with a minimum inhibitory concentration of 91.6 ± 28.8 µg/ml for A. alternata and F. oxysporum. Quantitative structure-activity relationship models revealed the octanolwater cleavage ratio as the molecular property, and the phenols as the main functional group contributing to antifungal activity. CONCLUSION: Terpenoids exhibit relevant antifungal activities that should be incorporated into the study of medicinal chemistry. Inclusion of in silico assays in the in vitro evaluation is a valuable tool in the search for and rational design of terpene derivatives as new potential antifungal agents.

Introducción: Los géneros Alternaria y Fusarium contienen especies patógenas para los humanos y los cultivos. Para su control, se han utilizado diversos antifúngicos. Sin embargo, su uso desmedido ha contribuido al desarrollo de agentes patógenos resistentes. Una alternativa para buscar y desarrollar nuevos agentes antimicóticos son los aceites esenciales y sus componentes principales, los cuales poseen diversas actividades biológicas de interés para la medicina y en la preservación de alimentos. Objetivo: Evaluar in vitro e in silico las actividades antifúngicas de terpenoides contra Alternaria alternata y Fusarium oxysporum. Materiales y métodos: Se evaluaron in vitro las concentraciones inhibitorias mínimas y las concentraciones fungicidas mínimas de 27 constituyentes de aceites esenciales contra A. alternata y F. oxysporum. Además, mediante algoritmos genéticos, se crearon modelos cuantitativos de la relación estructura-actividad para determinar las propiedades estructurales y fisicoquímicas relacionadas con la actividad antifúngica. Resultados: Los compuestos evaluados mostraron ser antifúngicos activos. El timol fue el compuesto con mayor actividad, con un valor de concentración inhibitoria mínima de 91.6 ± 28.8 µg/ml, tanto para Alternaria alternata como para Fusarium oxysporum. Los modelos cuantitativos de la relación estructura-actividad incluyeron la avidez por los lípidos y los fenoles como los principales grupos funcionales que contribuyen en la actividad antifúngica. Conclusión: Los terpenoides poseen actividades antifúngicas relevantes para ser incorporados en el estudio de la química medicinal. La inclusión de pruebas in silico a la evaluación in vitro es una herramienta útil para la búsqueda y el diseño racional de derivados terpénicos como posibles agentes antifúngicos.

SRAP markers as an alternative tool for Alternaria classification.

Alternaria is one of the main fungal contaminants of cereal grains worldwide with the potential to produce mycotoxins hazardous to human and animal health. Many studies have been carried out to characterize Alternaria sp.-grp. using traditional morphology or polyphasic approach, but a good correlation between morphological sp.-grp., molecular, and chemotaxonomic groups has not always been achieved. For this reason, this study aimed to investigate the usefulness of a cheaper alternative tool, SRAP markers, in identifying Alternaria sp.-grps. obtained from Argentinean barley grains and to compare it with preliminary characterization using morphological traits, phylogeny, and metabolite profiles. Fifty-three Alternaria isolates from barley grains of the main producing regions of Argentina were analyzed with four combinations of SRAP markers. The UPGMA dendrogram, based on the Simple Matching similarity coefficient, revealed three distinct groups. SRAP markers allowed the separation of Alternaria from Infectoriae sections in agreement with the results of a polyphasic approach previously made. Besides, isolates of A. arborescens sp.-grp. were clustered in a separate group from isolates of A. tenuissima and A. alternata sp.-grp., which were grouped in the same cluster. SRAP markers are a recommended tool for classifying Alternaria isolates because of its simplicity, reliability, and cost-effectiveness compared to other molecular markers.

First Report of Alternaria alternata Causing Leaf Spot on Broccoli in Ecuador.

In Ecuador, broccoli (Brassica oleracea var. italica) production is located in the Andean region, specifically Cotopaxi-Ecuador (INEC, 2019). A leaf pathogen has been constantly observed in this area, showing brown circular necrosis surrounded by yellowish halo-like spots causing leaf death (Fig. 1a). This pathogen was believed to be Alternaria sp.; however, the species was not determined either using classical or molecular tools. In 2021, ten leaves showing similar symptoms were collected in Cotopaxi and sent for pathogen identification. Here, leaf explants (0.25 cm2) showing disease symptoms were surface sterilized with 2% sodium hypochlorite (NaClO) and 70% ethanol (C2H6O), rinsed with sterile water, and transferred to Potato Dextrose Agar (PDA) media. Petri dishes were incubated in darkness at 25°C for five days. The single hyphal tip method was used to purify the cultures on PDA. Fifteen pure isolates were obtained after incubating for 14 days. Isolates were incubated under blacklight for two days to induce fungal sporulation. All isolates presented early white cotton-like mycelium that later became dark green (Fig 1b). Under the microscope, we observed straight primary conidia in simple or branched chains. Also, the conidia were obclavate, long ellipsoids, moderate in size (19.5-43.9 µm in length, 7.1-17.2 µm in width), and septate with few longitudinal septa. Lastly, the conidium body can narrow itself into a secondary conidia (Fig 1c) (Woudenberg et al., 2013). According to colony and conidia morphology, isolates were identified as Alternaria sp. (Woudenberg et al., 2013). Five isolates were randomly selected for DNA extraction and sequencing of ITS (internal transcribed spacer; Chou, H.H. and Wu, W.S. 2002), TEF (translation elongation factor; O'Donnell et al., 1998), and RPB2 (RNA polymerase II second largest subunit; Liu et al., 1999) gene regions. DNA sequences obtained from each marker were identical for all isolates. Consensus sequences and alignment were built using ClustalX in MEGA X (Kumar et al., 2018). Consensus sequences were deposited in GenBank with the following accession numbers: ITS, ON982232; TEF, ON983964; RPB2, ON983963. A multilocus Bayesian inference phylogenetic tree was constructed in Beast software (version 1.8.4) using the concatenated sequences (Drummond et al., 2012; Maharachchikumbura et al., 2014). The isolates in our study clustered with isolates of Alternaria alternata, confirming their identity (Figure 2). For Koch's postulates, healthy broccoli plants were grown in sterile soil for six weeks. The fungal conidia were suspended in sterile distilled water (1×106 conidia/ml), and the leaves were inoculated by spraying the spore solution. The control treatment was sprayed with sterile distilled water alone. Plants were maintained at 28°C and had more than 85% relative humidity (Sigillo et al., 2020). Seven days after inoculation, plants showed chlorosis and necrosis. Ten days later, 100% of the treated leaves presented brown circular necrosis (Fig. 1d). Control plants showed no disease symptoms. Re-isolation of the pathogen from the diseased leaf tissue was performed as previously described. The isolates presented the exact morphology of pure cultures obtained from field-diseased leaves. The pathogenicity test was performed twice. To our knowledge, this is the first report on A. alternata being the causal agent of leaf spot on broccoli in Ecuador. Disease diagnosis contributes to providing strategies against this pathogen. Further investigations are needed to find biological/chemical techniques or cultivar resistance to control this pathogen in broccoli.

Interacting Abiotic Factors Affect Growth and Mycotoxin Production Profiles of Alternaria Section Alternaria Strains on Chickpea-Based Media.

Chickpea is susceptible to fungal infection and mycotoxin contamination. Argentina exports most of its chickpea production; thus, its quality is of concern. The Alternaria fungal genus was found to be prevalent in chickpea samples from Argentina. The species within this genus are able to produce mycotoxins, such as alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TA). In this context, we evaluated the effect of water activity (0.99, 0.98, 0.96, 0.95, 0.94, 0.92, and 0.90 aW), temperature (4, 15, 25, and 30 °C), incubation time (7, 14, 21, and 28 days), and their interactions on mycelial growth and AOH, AME, and TA production on chickpea-based medium by two A. alternata strains and one A. arborescens strain isolated from chickpea in Argentina. Maximum growth rates were obtained at the highest aW (0.99) and 25 °C, with growth decreasing as the aW of the medium and the temperature were reduced. A. arborescens grew significantly faster than A. alternata. Mycotoxin production was affected by both variables (aW and temperature), and the pattern obtained was dependent on the strains/species evaluated. In general, both A. alternata strains produced maximum amounts of AOH and AME at 30 °C and 0.99-0.98 aW, while for TA production, both strains behaved completely differently (maximum levels at 25 °C and 0.96 aW for one strain and 30 °C and 0.98 aW for the other). A. arborescens produced maximum amounts of the three toxins at 25 °C and 0.98 aW. Temperature and aW conditions for mycotoxin production were slightly narrower than those for growth. Temperature and aW conditions assayed are those found during chickpea grain development in the field, and also could be present during storage. This study provides useful data on the conditions representing a risk for contamination of chickpea by Alternaria toxins.

Chenopodium quinoa, a New Host for Alternaria Section Alternata and Alternaria Section Infectoriae Causing Yellow Leaf Blotch Disease.

Quinoa (Chenopodium quinoa Willd.) is a native American crop mainly grown in the Andes of Bolivia and Peru. During the last decades, the cultivation of quinoa has expanded to more than 125 countries. Since then, several diseases of quinoa have been characterized. A leaf disease was observed on quinoa plants growing in an experimental plot in Eastern Denmark in 2018. The symptoms produced by the associated fungi consisted of small yellow blotches on the upper surface of leaves with a pale chlorotic halo surrounding the lesion. These studies used a combination of morphology, molecular diagnostics, and pathogenicity tests to identify two different Alternaria species belonging to Alternaria sections Infectoriae and Alternata as the causal agent of observed disease symptoms. To the best of our knowledge, this is the first report of Alternaria spp. as foliar pathogens of quinoa. Our findings indicate the need for additional studies to determine potential risks to quinoa production.

Antifungal activity and aroma persistence of free and encapsulated Cinnamomum cassia essential oil in maize.

The objective of this study was to evaluate the chemical composition and antifungal activity of free and encapsulated Cinnamomum cassia essential oil (EO) against Penicillium crustosum, Alternaria alternata, and Aspergillus flavus, and the aroma persistence in maize flour. Trans-cinnamaldehyde (TC) was identified as the major compound (86 %) in the C. cassia EO. The EO was encapsulated by spray-dryer with 45.26 % efficiency using gum arabic (GA) and maltodextrin (MD) in a ratio of 1:1 (m/m). C. cassia EO showed antifungal activity against A. alternata, A. flavus, and P. crustosum, with a minimum inhibitory concentration (MIC) of 0.5 % for both free and standard TC, and 5 % for the encapsulated EO. Fungal growth inhibition was evaluated under exposition to vapors at different concentrations of C. cassia EO and TC standard, with MIC of 6 % and 8 % against P. crustosum, 4 % and 1 % A. alternata, and 4 % A. flavus, respectively. The sensory analysis results of the free and encapsulated C. cassia EO in maize flour showed a significant difference between the treated samples in relation to the standard sample (p < 0.05). The sample with free EO has high aroma intensity persistence, while the samples treated with encapsulated EO were evaluated as being closer to the standard sample. The results suggest that the encapsulated C. cassia EOs can be used as natural alternatives to control fungi in maize flour.

In vitro and Quantitative and Structure Activity Relationship (QSAR) evaluation of the antifungal activity of terpenoid constituents of essential oils against Alternaria alternata and Fusarium oxysporum / Evaluación in vitro y QSAR (Quantitative and Structure-Activity Relationship) de la actividad antifúngica de terpenoides obtenidos de aceites esenciales frente a Alternaria alternata y Fusarium oxysporum

Introducción. Los géneros Alternaria y Fusarium contienen especies patógenas para los humanos y los cultivos. Para su control, se han utilizado diversos antifúngicos. Sin embargo, su uso desmedido ha contribuido al desarrollo de agentes patógenos resistentes. Una alternativa para buscar y desarrollar nuevos agentes antimicóticos son los aceites esenciales y sus componentes principales, los cuales poseen diversas actividades biológicas de interés para la medicina y en la preservación de alimentos. Objetivo. Evaluar in vitro e in silico las actividades antifúngicas de terpenoides contra Alternaria alternata y Fusarium oxysporum. Materiales y métodos. Se evaluaron in vitro las concentraciones inhibitorias mínimas y las concentraciones fungicidas mínimas de 27 constituyentes de aceites esenciales contra A. alternata y F. oxysporum. Además, mediante algoritmos genéticos, se crearon modelos cuantitativos de la relación estructura-actividad para determinar las propiedades estructurales y fisicoquímicas relacionadas con la actividad antifúngica. Resultados. Los compuestos evaluados mostraron ser antifúngicos activos. El timol fue el compuesto con mayor actividad, con un valor de concentración inhibitoria mínima de 91.6 ± 28.8 pg/ml, tanto para Alternarla alternata como para Fusarium oxysporum. Los modelos cuantitativos de la relación estructura-actividad incluyeron la avidez por los lípidos y los fenoles como los principales grupos funcionales que contribuyen en la actividad antifúngica. Conclusión. Los terpenoides poseen actividades antifúngicas relevantes para ser incorporados en el estudio de la química medicinal. La inclusión de pruebas in silico a la evaluación in vitro es una herramienta útil para la búsqueda y el diseño racional de derivados terpénicos como posibles agentes antifúngicos.

Introduction. Fungal genera Alternaría and Fusarium include human and plant pathogenic species. Several antifungals have been used for their control, hut excessive use has contributed to resistance development in pathogens. An alternative to searching for and developing new antifungal agents is using essential oils and their main components, which have biological activities of interest in medicine and food production. Objective. To evaluate in vitro and in silico the antifungal activities of terpenoids against Alternaria alternata and Fusarium oxysporum. Materials and methods. The minimum inhibitory concentration and minimum fungicidal concentration values of 27 constituents of essential oils used against Alternaria alternata and Fusarium oxysporum were evaluated in vitro. In addition, using genetic algorithms, quantitative models of the structure-activity relationship were used to identify the structural and physicochemical properties related to antifungal activity. Results. The evaluated compounds proved to be effective antifungals. Thymol was the most active with a minimum inhibitory concentration of 91.6 ± 28.8 pg/ml for A. alternata and F. oxysporum. Quantitative structure-activity relationship models revealed the octanol-water cleavage ratio as the molecular property, and the phenols as the main functional group contributing to antifungal activity. Conclusion. Terpenoids exhibit relevant antifungal activities that should be incorporated into the study of medicinal chemistry. Inclusion of in silico assays in the in vitro evaluation is a valuable tool in the search for and rational design of terpene derivatives as new potential antifungal agents.

Activity of Aqueous Extracts from Native Plants of the Yucatan Peninsula against Fungal Pathogens of Tomato In Vitro and from Croton chichenensis against Corynespora cassiicola on Tomato.

Plant extracts are a valuable alternative to control pathogens of horticultural crops. In the present study, four species of pathogenic fungi were isolated from leaf spots on Solanum lycopersicum and identified by traditional and molecular techniques as Alternaria alternata ITC24, Corynespora cassiicola ITC23, Curvularia lunata ITC22, and Fusarium equiseti ITC32. When 11 aqueous extracts from eight native plants of the Yucatan Peninsula were tested against the four fungi in vitro, the extract from Croton chichenensis roots was most active, inhibiting mycelial growth (79-100%), sporulation (100%), and conidial germination (71-100%) at 3% (w/v). A logarithmic-diagrammatic scale of the pathosystem C. cassiicola-S. lycopersicum was established and used to assess disease severity on inoculated tomato plants in a greenhouse after treatment with the aqueous extract from C. chichenensis roots at 12% (w/v). After 21 days, the disease severity was 57% lower than on the control without extract applied. This dose of the extract was not phytotoxic to tomato leaves and was compatible with the beneficial organisms Bacillus subtilis CBCK47 and Trichodema asperellum Ta13-17. The antifungal efficacy of C. chichenensis is highly promising for incorporation into integrated disease management of tomato crops.

Dual-transcriptomic datasets evaluating the effect of the necrotrophic fungus Alternaria brassicicola on Arabidopsis germinating seeds.

Many fungal pathogens are carried and transmitted by seeds. These pathogens affect germination and seed quality. Their transmission from the germinating seed to seedling causes many diseases in crops. Seed defense mechanisms during germination are poorly documented. RNA-seq experiments were used to describe the molecular mechanisms involved in seed interaction with a necrotrophic fungus. Here the Arabidopsis thaliana/Alternaria brassicicola pathosystem was used to perform dual-transcriptomic approach. Arabidopsis thaliana seeds and necrotrophic fungus transcripts were identified at critical germination and seedling establishment stages. Total RNA was extracted from healthy and infected germinating seeds and seedlings at 3, 6 and 10 days after sowing. Transcript libraries were made and sequenced, then fungal and plant short reads were mapped and quantified respectively against Arabidopsis thaliana and Alternaria brassicicola reference transcriptomes. This dual-transcriptomic approach revealed that 3409, 7506 and 8589 Arabidopsis thaliana genes showed a differential expression at respectevely 3, 6 and 10 days after sowing between healthy and infected seeds, including 1192 genes differentially expressed at the three studied stages. Moreover, in this experiement, we also identified the dynamic of the transcript changes occurring at the same stages in the necrotrophic fungus concomitantly during germination and seedling establishment.

Evaluating the potential of Pediococcus pentosaceus as a biocontrol agent against tenuazonic acid-producing Alternaria alternata on livestock feeds.

AIMS: This study aims to demonstrate the potential of the lactic acid bacteria (LAB) Pediococcus pentosaceus LBM18 against the mycotoxin-producing Alternaria alternata TEF-1A and highlight its application as an effective grain silage inoculant to control mycotoxin contamination. METHODS AND RESULTS: The antifungal properties of Ped. pentosaceus lyophilized (PPL) were assessed by evaluating its effect on A. alternata TEF-1A grown in a corn silage-based medium, which included morphological changes by Scanning Electron Microscopy (SEM) observations, growth rate, conidia production assays, and inhibition of Tenuazonic acid (TeA) production by high-performance liquid chromatography (HPLC-MS/MS) analyses. Furthermore, TeA biosynthesis was monitored for changes at the molecular level by PKS gene expression. The growth and sporulation processes of A. alternata TEF-1A were affected by Ped. pentosaceus LBM18 in a concentration-dependent manner. Moreover, a significant inhibition of TeA production (74.3%) and the transcription level of the PKS gene (42.9%) was observed. CONCLUSIONS: Ped. pentosaceus is one of the promising LAB to be applied as an inoculant for corn silage preservation, aiming to inhibit mycotoxigenic fungi growth and their mycotoxin production. SIGNIFICANCE AND IMPACT OF THE STUDY: Ped. pentosaceus could be used as an inoculant to reduce fungal and mycotoxins contamination in grain silage production.

Morphological and chemical characterization of Alternaria populations from apple fruit.

Alternaria is a frequent contaminant of apple fruit, causing severe economic losses. It can produce external lesions and mouldy core, characterised by a rotten area in the apple core. In the present study, morphological and chemical characterization of Alternaria from apples was performed, evaluating differences related to agricultural practices and type of disease. A low morphological diversity was observed; most of the isolates were identified as A. tenuissima sp.-grp. (95 %). A. arborescens sp.-grp. and A. gaisen sp.-grp. were present in a proportion of 1 %, and 3 % of the isolates showed intermediate characteristics between these sp.-grps. and were identified as Alternaria sp. The chemical diversity was greater; 27 secondary metabolites were produced by the apple isolates. The most frequents were altertoxin-I (85 %), altechromone A (76 %), tentoxin (69 %), and tenuazonic acid (68 %). The alternariols were produced in a lower frequency when comparing with isolates from other crops; alternariol, 58 % and alternariol monomethyl ether, 57 %. The predominant secondary metabolite profile included compounds from different chemical families, such as dibenzopyrones, tetramic acids, perylene quinones, and cyclic tetrapeptides. A wider metabolomic capacity was observed in isolates from conventional apples when compared to those from organic fruit, with the predominance of strong producers of altertoxins and alternariols. The isolates from mouldy core showed higher ability to produce metabolites from different chemical families than those from external lesions. The wide chemical diversity of the Alternaria apple population should be considered to assess the health risk associated with apple by-products.

Seed Transmission of Pathogens: Non-Canonical Immune Response in Arabidopsis Germinating Seeds Compared to Early Seedlings against the Necrotrophic Fungus Alternaria brassicicola.

The transmission of seed-borne pathogens by the germinating seed is responsible for major crop diseases. The immune responses of the seed facing biotic invaders are poorly documented so far. The Arabidopsis thaliana/Alternaria brassicicola patho-system was used to describe at the transcription level the responses of germinating seeds and young seedling stages to infection by the necrotrophic fungus. RNA-seq analyses of healthy versus inoculated seeds at 3 days after sowing (DAS), stage of radicle emergence, and at 6 and 10 DAS, two stages of seedling establishment, identified thousands of differentially expressed genes by Alternaria infection. Response to hypoxia, ethylene and indole pathways were found to be induced by Alternaria in the germinating seeds. However, surprisingly, the defense responses, namely the salicylic acid (SA) pathway, the response to reactive oxygen species (ROS), the endoplasmic reticulum-associated protein degradation (ERAD) and programmed cell death, were found to be strongly induced only during the latter post-germination stages. We propose that this non-canonical immune response in early germinating seeds compared to early seedling establishment was potentially due to the seed-to-seedling transition phase. Phenotypic analyses of about 14 mutants altered in the main defense pathways illustrated these specific defense responses. The unexpected germination deficiency and insensitivity to Alternaria in the glucosinolate deficient mutants allow hypothesis of a trade-off between seed germination, necrosis induction and Alternaria transmission to the seedling. The imbalance of the SA and jasmonic acid (JA) pathways to the detriment of the JA also illustrated a non-canonical immune response at the first stages of the seedling.

Native Cultivable Bacteria from the Blueberry Microbiome as Novel Potential Biocontrol Agents.

Blueberry production is affected by fungal postharvest pathogens, including Botrytis cinerea and Alternaria alternata, the causative agents of gray mold disease and Alternaria rot, respectively. Biocontrol agents adapted to blueberries and local environments are not known to date. Here, we report on the search for and the identification of cultivable blueberry epiphytic bacteria with the potential to combat the aforementioned fungi. Native, blueberry-borne bacterial strains were isolated from a plantation in Tucumán, Argentina and classified based on 16S rRNA gene sequences. Antagonistic activities directed at B. cinerea and A. alternata were studied in vitro and in vivo. The 22 bacterial strains obtained could be attributed to eleven different genera: Rosenbergiella, Fictibacillus, Bacillus, Pseudomonas, Microbacterium, Asaia, Acinetobacter, Curtobacterium, Serratia, Sphingomonas and Xylophilus. Three strains displaying antagonistic impacts on the fungal pathogens were identified as Bacillus velezensis (BA3 and BA4) and Asaia spathodeae (BMEF1). These strains are candidates for biological control agents of local blueberry production and might provide a basis for the development of eco-friendly, sustainable alternatives to synthetic pesticides.

Screening of Endophytic Bacteria Isolated from Weed Plant to Biocontrol Stem Rot Disease on Pitaya (Hylocereus undatus)

Abstract Endophytic bacteria from weed are emerging as valuable alternatives for biochemical pesticides in sustainable agriculture. This study aims to discover the antagonistic effects of some endophytic bacteria isolated from weed (Lactuca indica) against A. alternata, a casual of stem end rot disease of pitaya. A total of 14 endophytic bacteria were isolated and four of them presented in vitro antagonistic activity against A. alternata. Of four, strain L115 significantly inhibited the pathogenic growth with a mean inhibition diameter of 12.67 ± 0.02 mm, while the other three (strain L110, L111, and L114) showed a weak inhibition. The results indicated that strain L115 could belong to the Bacillaceae family while the other three (strain L110, L111, and L114) had characteristics of the Pseudomonadaceae family. Interestingly, strain L115 showed positive results for phosphate solubilization, indole acetic acid (IAA), and biofilm production, whereas three other strains presented weak capabilities for phosphate solubilization, biofilm production and IAA production. In addition, the filtrate of strain L115 presented antifungal activity on biocontrol tests in vitro. Especially, strain L115 significantly increased seedling biomass of pitaya and tomato compared to the control. Hence, these results suggest strain L115 has the potential to be used as biocontrol agents against A. alternata. More studies should be done in the future to evaluate their efficiency in field conditions.