Multi-Locus Phylogenetic Analysis Revealed the Association of Six Colletotrichum Species with Anthracnose Disease of Coffee (Coffea arabica L.) in Saudi Arabia.

Several Colletotrichum species are able to cause anthracnose disease in coffee (Coffea arabica L.) and occur in all coffee production areas worldwide. A planned investigation of coffee plantations was carried out in Southwest Saudi Arabia in October, November, and December 2022. Various patterns of symptoms were observed in all 23 surveyed coffee plantations due to unknown causal agents. Isolation from symptomatic fresh samples was performed on a PDA medium supplemented with streptomycin sulfate (300 mg L-1) and copper hydroxide (42.5 mg L-1). Twenty-seven pure isolates of Colletotrichum-like fungi were obtained using a spore suspension method. The taxonomic placements of Colletotrichum-like fungi were performed based on the sequence dataset of multi-loci of internal transcribed spacer region rDNA (ITS), chitin synthase I (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), ß-tubulin (TUB2), and partial mating type (Mat1-2) (ApMat) genes. The novel species are described in detail, including comprehensive morphological characteristics and colored illustrations. The pathogenicity of the isolated Colletotrichum species was assessed on detached coffee leaves as well as green and red fruit under laboratory conditions. The multi-locus phylogenetic analyses of the six-loci, ITS, ACT, CHS-1, TUB2, GAPDH and ApMat, revealed that 25 isolates were allocated within the C. gloeosporioides complex, while the remaining two isolates were assigned to the C. boninense complex. Six species were recognized, four of them, C. aeschynomenes, C. siamense, C. phyllanthi, and C. karstii, had been previously described. Based on molecular analyses and morphological examination comparisons, C. saudianum and C. coffeae-arabicae represent novel members within the C. gloeosporioides complex. Pathogenicity investigation confirmed that the Colletotrichum species could induce disease in coffee leaves as well as green and red fruits with variations. Based on the available literature and research, this is the first documentation for C. aeschynomenes, C. siamense, C. karstii, C. phyllanthi, C. saudianum, and C. coffeae-arabicae to cause anthracnose on coffee in Saudi Arabia.

Mycotoxins from Tomato Pathogenic Alternaria alternata and Their Combined Cytotoxic Effects on Human Cell Lines and Male Albino Rats.

The Alternaria species are considered to produce a plethora of several mycotoxins constituting a risk factor for both human and animal health. This work aimed mainly to explore the cytotoxicity of a combined mixture of altenuene (ALT), alternariol (AOH), tenuazonic acid (TeA), and altenuisol (AS) toxins produced by pathogenic A. alternata toward human oral epithelial cells (PCS-200-014), lung fibroblast cells (WI-38), and male albino rats. The sequencing of the multi-locus, RNA polymerase second largest subunit (rpb2), glyceraldehyde-3-phosphate dehydrogenase (gapdh), and Alternaria major allergen gene (Alt a 1) was performed to infer relationships among isolated Alternaria species. The phylogenetic analysis of gapdh, rpb2, and Alt-a 1 sequence data indicated that all isolates resided in A. alternata. The pathogenic potentiality of A. alternata was investigated on tomato plants cv. super strain B under greenhouse conditions, and all isolates were pathogenic to tomato plants, with significant (p < 0.05) variations. The ability of A. alternata isolates to produce mycotoxins was also explored using high-performance liquid chromatography (HPLC). All tested isolates were able to produce at least one of the assessed mycotoxins-ALT, AOH, TeA, and AS-and ALT was reported as the dominant mycotoxin, produced by 80% of A. alternata isolates. The cytotoxic properties of the combined mixture of ALT, AOH, TeA, and AS at concentrations of 31.25, 62.50, 125, 250, and 500 µg/mL were assessed via the MTT assay method after exposure for 24 h versus the control. The treatment of both cell lines with combined mixtures of ALT, AOH, TeA, and AS showed a dose-dependent decrease in cell viability. The highest concentrations tested at 62.50, 125, 250, and 500 µg/mL significantly decreased cell viability and caused cell damage compared to the lowest concentration of 31.25 µg/mL and the control. The cytotoxicity and genotoxicity of the combined mixtures of ALT, AOH, TeA, and AS on male albino rats were also investigated via the gene expression of (TNF-α) and using hematological (CBC), chemical (alanine aminotransferase (ALT), aspartate aminotransferase (AST) and urea and creatinine), and histopathological analyses. A marked increase was observed in the levels of ALT, AST, urea and creatinine, TNF-α gene expression, red blood cells (RBCs), white blood cells (WBCs), hemoglobin (Hb), and packed cell volume % (PCV) after 28 days of exposure relative to the untreated control. Pathological alterations were also observed in the liver and kidney tissues of rats. Conclusively, this work provides a new understanding on the cytotoxicity and genotoxicity of mycotoxins of pathogenic A. alternata from tomatoes.

Diversity among Lasiodiplodia Species Causing Dieback, Root Rot and Leaf Spot on Fruit Trees in Egypt, and a Description of Lasiodiplodia newvalleyensis sp. nov.

Lasiodiplodia (family Botryosphaeriaceae) is a widely distributed fungal genus that causes a variety of diseases in tropical and subtropical regions. During 2020−2021, a routine survey of fruit tree plants was conducted in five Egyptian Governorates, and fresh samples exhibiting dieback, decline, leaf spot and root rot symptoms were collected. Collection from eight different symptomatic leaves, twigs, branches and roots of fruit trees yielded 18 Lasiodiplodia-like isolates. The sequencing data from the internal transcribed spacer region (ITS), partial translation elongation factor 1-alpha (tef1-a) and ß-tubulin (tub2) were used to infer phylogenetic relationships with known Lasiodiplodia species. Two isolates obtained from black necrotic lesions on Phoenix dactylifera leaves were identified as a putative novel species, L. newvalleyensis sp. nov., and were thus subjected to further morphological characterization. The results of isolation and molecular characterization revealed that L. theobromae (n = 9) was the most common species on Mangifera indica, Citrus reticulata, C. sinensis, Ficus carica, Prunus persica, Prunus armeniaca and Pyrus communis trees. Lasiodiplodia pseudotheobromae (n = 5) was isolated from M. indica, Prunus persica and C. sinensis. Lasiodiplodia laeliocattleyae (n = 2) was isolated from C. reticulata. Pathogenicity test results suggested that all Lasiodiplodia species were pathogenic to their hosts. The present study is considered the first to characterize and decipher the diversity of Lasiodiplodia species associated with fruit trees in Egypt, using the multi-locus ITS, tef1-a and tub2 sequence data, along with morphological and pathogenic trials. To our knowledge, this is the first report of L. newvalleyensis on Phoenix dactylifera and L. laeliocattleya on C. reticulata in Egypt and worldwide.

Studies on the efficacy of electrolysed oxidising water to control Aspergillus carbonarius and ochratoxin A contamination on grape.

Ochratoxin A (OTA) occurrence in grapes is caused by black Aspergilli (Aspergillus carbonarius followed by A. niger) vineyards contamination. It depends on climatic conditions, geographical regions, damage by insects, and grape varieties. Good agricultural practices, pesticides, and fungicides seem adequate to manage the problem during low OTA risk vintages, but the development of new strategies is always encouraged, especially when an extremely favourable condition occurs in the vineyard. Electrolysed oxidising water (EOW) has become an interesting alternative to chemicals in agriculture, mainly during the post-harvest phase. This study tested the fungicidal efficacy of EOW generated by potassium chloride, in vitro, on black Aspergilli conidia, and detached grape berries infected by A. carbonarius. Then, during field trials on Primitivo cv vineyard treated with EOW, A. carbonarius contamination, and OTA levels were compared with Switch® fungicide treatment (0.8 g/l). Black Aspergilli conidia were killed on plate assay after 2 min of treatment by EOW containing >0.4 g/l of active chlorine. EOW (0.6 g/l active chlorine) treatment reduced the rate of A. carbonarius infections in vitro of about 87-92% on detached berries and, more than half in the field trials, although Switch® showed better performance. A significant reduction in the OTA concentration was observed for the EOW and Switch® treatments in vitro (92% and 96%, respectively), while in the field trials, although the average decrease in OTA was recorded in the treated grapes, it was not statistically significant. These results highlighted that EOW could be considered effective, as a substitute for fungicides, to reduce the contamination of A. carbonarius and OTA on grapes.

Interacting climate change factors (CO2 and temperature cycles) effects on growth, secondary metabolite gene expression and phenotypic ochratoxin A production by Aspergillus carbonarius strains on a grape-based matrix.

Little is known on the impact that climate change (CC) may have on Aspergillus carbonarius and Ochratoxin A (OTA) contamination of grapes, especially in the Mediterranean region where in CC scenarios temperature are expected to increase by +2-5 °C and CO2 from 400 to 800/1200 ppm. This study examined the effect of (i) current and increased temperature in the alternating 11.5 h dark/12.5 h light cycle (15-28 °C vs 18-34 °C), representative of the North Apulia area, South Italy and (ii) existing and predicted CO2 concentrations (400 vs 1000 ppm), on growth, expression of biosynthetic genes (AcOTApks, AcOTAnrps, AcOTAhal, AcOTAp450, AcOTAbZIP) and regulatory genes of Velvet complex (laeA/veA/velB, "velvet complex") involved in OTA biosynthesis and OTA phenotypic production by three strains of A. carbonarius. The experiments made on a grape-based matrix showed that elevated CO2 resulted in a general stimulation of growth and OTA production. These results were also supported by the up-regulation of both structural and regulatory genes involved in the OTA biosynthesis. Our work has shown for the first time that elevated CO2 concentration in the Mediterranean region may result in an increased risk of OTA contamination in the wine production chain.

Comparative Genomic Analysis of Ochratoxin A Biosynthetic Cluster in Producing Fungi: New Evidence of a Cyclase Gene Involvement.

The widespread use of Next-Generation Sequencing has opened a new era in the study of biological systems by significantly increasing the catalog of fungal genomes sequences and identifying gene clusters for known secondary metabolites as well as novel cryptic ones. However, most of these clusters still need to be examined in detail to completely understand the pathway steps and the regulation of the biosynthesis of metabolites. Genome sequencing approach led to the identification of the biosynthetic genes cluster of ochratoxin A (OTA) in a number of producing fungal species. Ochratoxin A is a potent pentaketide nephrotoxin produced by Aspergillus and Penicillium species and found as widely contaminant in food, beverages and feed. The increasing availability of several new genome sequences of OTA producer species in JGI Mycocosm and/or GenBank databanks led us to analyze and update the gene cluster structure in 19 Aspergillus and 2 Penicillium OTA producing species, resulting in a well conserved organization of OTA core genes among the species. Furthermore, our comparative genome analyses evidenced the presence of an additional gene, previously undescribed, located between the polyketide and non-ribosomal synthase genes in the cluster of all the species analyzed. The presence of a SnoaL cyclase domain in the sequence of this gene supports its putative role in the polyketide cyclization reaction during the initial steps of the OTA biosynthesis pathway. The phylogenetic analysis showed a clustering of OTA SnoaL domains in accordance with the phylogeny of OTA producing species at species and section levels. The characterization of this new OTA gene, its putative role and its expression evidence in three important representative producing species, are reported here for the first time.

Effects of temperature and water activity change on ecophysiology of ochratoxigenic Aspergillus carbonarius in field-simulating conditions.

Ochratoxin A (OTA) is the primary mycotoxin threat in wine and dried vine fruits. Its presence in grape and wine is strongly related to climatic conditions and the expected climate change could represent a risk of increasing fungal colonization and OTA contamination in grapes. In this regard, the interacting effect of i) different conditions of water availability (0.93 and 0.99aw) and ii) different 10 h/14 h dark/light alternating temperature conditions simulating a nowadays (18/31 °C) and climate change scenario (20/37 °C) in high OTA risk areas of Apulia region, were studied. Lag phases prior to growth, mycelial growth rate, the expression of biosynthesis, transcription factors and regulatory genes of OTA cluster and OTA production were analysed in Aspergillus carbonarius ITEM 5010 under the combined effect of different climatic factors. At 18/31 °C and under water stress conditions (0.93 aw) the growth rate was slower than at 0.99 aw; on the contrary, at 20/37 °C a higher growth rate was observed at 0.93 aw. An over-expression of OTA genes and genes belonging to the global regulator Velvet complex was observed at 18/31 °C and 0.99 aw, with the specific OTA pathway transcription factor bZIP showing the highest expression level. The up-regulated transcription profile of the genes positively correlated with OTA production higher at 18/31 °C than at 20/37 °C and 0.99 aw; while no OTA production was detected at 0.93 aw at each of the temperature conditions tested. These findings provide preliminary evidence that the possible increase of the temperature, likely to happen in some areas of the Apulia region, may results in a reduction of both A. carbonarius spoilage and OTA production in grapes.

Cultivar Resistance against Colletotrichum asianum in the World Collection of Mango Germplasm in Southeastern Brazil.

During the spring of 2014, a wide survey was conducted in one of the most important mango (Mangifera indica) cultivating areas located in Minas Gerais State (Brazil) to ascertain the causal agent of severe anthracnose infections and to evaluate disease susceptibility within a world collection of mango germplasm. Overall, 86 cultivars were monitored and 152 fungal isolates recovered from infected samples were identified by morphological characterization, DNA sequencing and phylogenetic analyses. All isolates were identified as Colletotrichum asianum. Under natural disease pressure, it has been possible to ascertain a variable tolerance degree within the germplasm collection. By applying a categorized classification, cultivars were classified as follows: 10 highly sensitive (11.6%), 13 sensitive (15.1%), 18 moderately sensitive (20.9%), 23 moderately tolerant (26.7%), 11 tolerant (12.8%), and 11 highly tolerant (10.4%). The most susceptible cultivars to anthracnose were Ubà, Quinzenga, Amarelinha da Sementeira followed by Aroeira and Correjo, whereas Mallika followed by Ourinho and Lita resulted in the least susceptible cultivars. To the authors' knowledge, this is the first large-scale evaluation of mango susceptibility to C. asianum infections within a wide number of cultivars. Anthracnose is a serious threat to mango production and assessment of cultivar response to disease could be useful in breeding programs.

Study of gene expression and OTA production by Penicillium nordicum during a small-scale seasoning process of salami.

Penicillium nordicum, an important and consistent producer of ochratoxin A (OTA), is a widely distributed contaminant of protein rich food with elevated NaCl. It is usually found on dry-cured meat products and is considered the main species responsible for their contamination by OTA. The aim of this work was to study the gene expression of a polyketide synthase (otapksPN) involved in P. nordicum OTA biosynthesis, and OTA production during a small-scale seasoning process. Fresh pork sausages were surface inoculated with P. nordicum and seasoned for 30days. Gene expression and OTA production were monitored throughout the seasoning process after 4, 5, 6, 7, 10, 14, and 30days. The expression of otapksPN gene was already detected after 4days and increased significantly after 7days of seasoning, reaching the maximum expression level after 10days (1.69×10(4)copies/100mg). Consistently with gene expression monitoring, OTA was detected from the 4th day and its content increased significantly from the 7th day, reaching the maximum level after 10days. In the late stages of the seasoning process, OTA did not increase further and the number of gene copies was progressively reduced after 14 and 30days.