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.

Isolation and Characterization of a Novel Pathogenesis-Related Protein-1 Gene (AvPR-1) with Induced Expression in Oat (Avena sativa L.) during Abiotic and Hormonal Stresses.

Pathogenesis-related protein-1 (PR-1) plays crucial roles in regulating plant responses to biotic and abiotic stresses. This study aimed to isolate and characterize the first PR-1 (AvPR-1) gene in oat (Avena sativa L.). AvPR-1 presented conserved signal peptide motifs and core amino acid composition in the functional protein domains as the protein sequence of AvPR-1 presented 98.28%, 97.7%, and 95.4% identity with known PR1 proteins isolated from Triticum aestivum PRB1-2-like, Triticum dicoccoides PRB1-2-like, and Aegilops tauschii subsp. tauschii, respectively. Bioinformatic analysis showed that the AvPR-1 protein belongs to the CAP superfamily (PF00188). Secondary and 3D structure analyses of the AvPR-1 protein were also conducted, confirming sequence conservation of PR-1 among studied species. The AvPR-1 protein harbors a calmodulin-binding domain located in its C-terminal part as previously shown for its wheat homolog TdPR1.2. Moreover, gene expression analysis showed that AvPR-1 was induced in response to many abiotic and hormonal stresses especially in leaves after treatment for 48 h. This is the first study exhibiting the expression profiles of the AvPR-1 gene under different stresses in oat.

Characterization and Control of Thielaviopsis punctulata on Date Palm in Saudi Arabia.

Date palm (Phoenix dactylifera L.) is the most important edible fruit crop in Saudi Arabia. Date palm cultivation and productivity are severely affected by various fungal diseases in date palm-producing countries. In recent years, black scorch disease has emerged as a devastating disease affecting date palm cultivation in the Arabian Peninsula. In the current survey, leaves and root samples were collected from deteriorated date palm trees showing variable symptoms of neck bending, leaf drying, tissue necrosis, wilting, and mortality of the entire tree in the Al-Ahsa region of Saudi Arabia. During microscopic examination, the fungus isolates growing on potato dextrose agar (PDA) media produced thick-walled chlamydospores and endoconidia. The morphological characterization confirmed the presence of Thielaviopsis punctulata in the date palm plant samples as the potential agent of black scorch disease. The results were further confirmed by polymerase chain reaction (PCR), sequencing, and phylogenetic dendrograms of partial regions of the ITS, TEF1-α, and ß-tubulin genes. The nucleotide sequence comparison showed that the T. punctulata isolates were 99.9-100% identical to each other and to the T. punctulata isolate identified from Iraq-infecting date palm trees. The pathogenicity of the three selected T. punctulata isolates was also confirmed on date palm plants of Khalas cultivar. The morphological, molecular, and pathogenicity results confirmed that T. punctulata causes black scorch disease in symptomatic date palm plants in Saudi Arabia. Furthermore, seven commercially available fungicides were also tested for their potential efficacy to control black scorch disease. The in vitro application of the three fungicides Aliette, Score, and Tachigazole reduced the fungal growth zone by 86-100%, respectively, whereas the in vivo studies determined that the fungicides Aliette and Score significantly impeded the mycelial progression of T. punctulata with 40% and 73% efficiency, respectively. These fungicides can be used in integrated disease management (IDM) strategies to curb black scorch disease.

High-Throughput Sequencing Identified Distinct Bipartite and Monopartite Begomovirus Variants Associated with DNA-Satellites from Tomato and Muskmelon Plants in Saudi Arabia.

The studies on the prevalence and genetic diversity of begomoviruses in Saudi Arabia are minimal. In this study, field-grown symptomatic tomato and muskmelon plants were collected, and initially, begomovirus infection was confirmed by the core coat protein sequences. Four tomato and two muskmelon plants with viral infections were further evaluated for Illumina MiSeq sequencing, and twelve sequences (2.7-2.8 kb) equivalent to the full-length DNA-A or DNA-B components of begomoviruses were obtained along with eight sequences (~1.3-1.4 kb) equivalent to the begomovirus-associated DNA-satellite components. Four begomovirus sequences obtained from tomato plants were variants of tomato yellow leaf curl virus (TYLCV) with nt sequence identities of 95.3-100%. Additionally, two tomato plants showed a mixed infection of TYLCV and cotton leaf curl Gezira virus (CLCuGeV), okra yellow crinkle Cameroon alphasatellite (OYCrCMA), and okra leaf curl Oman betasatellite (OLCuOMB). Meanwhile, from muskmelon plants, two sequences were closely related (99-99.6%) to the tomato leaf curl Palampur virus (ToLCPalV) DNA-A, whereas two other sequences showed 97.9-100% sequence identities to DNA-B of ToLCPalV, respectively. Complete genome sequences of CLCuGeV and associated DNA-satellites were also obtained from these muskmelon plants. The nt sequence identities of the CLCuGeV, OYCrCMA, and OLCuOMB isolates obtained were 98.3-100%, 99.5-100%, and 95.6-99.7% with their respective available variants. The recombination was only detected in TYLCV and OLCuOMB isolates. To our knowledge, this is the first identification of a mixed infection of bipartite and monopartite begomoviruses associated with DNA-satellites from tomato and muskmelon in Saudi Arabia. The begomovirus variants reported in this study were clustered with Iranian isolates of respective begomovirus components in the phylogenetic dendrogram. Thus, the Iranian agroecological route can be a possible introduction of these begomoviruses and/or their associated DNA-satellites into Saudi Arabia.