Isolation and Identification of Fungal Endophytes of the Cowpea in Khuzestan Province

Introduction: Endophytes are microorganisms that colonize internal tissues of plants without causing obvious symptoms. This study was conducted to isolate and identify endophytic fungi of the cowpea in Khuzestan province. Materials and methods: During 2016, eight healthy samples of the cowpea plants were collected from the important areas under cultivation in the northern Khuzestan province. The small parts of the roots, stems, leaves and pods were deeply surface sterilized for each samples and plated on Potato-Dextrose-Agar. Sixty fungal isolates obtained in this study were purified by single spore method. Based on morphological characteristics, 21 out of 60 isolates were selected for molecular study. The isolates were grown in Potato-Dextrose-Broth and mycelial biomass was recovered by passing through filter paper. DNA extraction was performed using a phenol- and chloroform- based organic method. The parts of the nrRNA gene (ITS and 28S-D1/D2 regions) were amplified using appropriate primer pair and then sequenced. Results: The isolates were analyzedon the basis of morphological characteristics in combination with BlASTn search algorithm and ITS sequence-based phylogeny. Accordingly, the isolates were identified as follows: Alternaria destruens, Alternaria sp., Curvularia mosaddeghii, Curvularia sp., Fusarium chlamydosporum, F. nygamai, F. falciforme, F. proliferatum, Fusarium sp. Macrophomina phaseolina and Penicillium oxalicum. Discussion and conclusion: Alternaria, Fusarium and Curvularia genera were the most abundant fungal endophytes into cowpea plants, growing in warm climate of the Khuzestan Province. To the best of our knowledge, this is the first study to report endophytic growth of A. destruens, Alternaria sp., M. phaseolina, F. chlamydosporum, F. nygamai, F. falciforme, F. proliferatum and P. oxalicum within cowpea plants

Effects of Imunit Insecticide on Biological Characteristics and Life Table Parameters of Spodoptera cilium (Lepidoptera: Noctuidae)

Imunit is a mixture of alpha-cypermethrin + teflubenzuron, and has been launched for controlling caterpillars. In this study, the effects of Imunit at LC50 and LC30 were investigated on parental and offspring generation of S. cilium, according to age-stage, two-sex life table. The experiments were conducted by leaf dipping method at 25 C and 60 5% relative humidity, under a cycle of 16 h fluorescent light and 8 h darkness. LC30 and LC50 concentrations of Imunit increased the immature developmental time of S. cilium in the offspring generation, while the LC50 of Imunit significantly reduced the developmental time of adults. The adult pre-oviposition period and total pre-oviposition period considerably increased when offspring were treated with LC50 of Imunit. In offspring of S. cilium exposed to LC50 and LC30 concentrations of Imunit, the gross reproductive rate (GRR), net reproduction rate (R0), the intrinsic rate of population increase (r), and the finite rate of population increase (l) significantly reduced compared to the control. This study showed that the application of Imunit at LC50 could suppress the S. cilium population and can be used in the integrated management program of this pest

Identification of Trichoderma Species Using Partial Sequencing of nrRNA and tef1α Genes with Report of Trichoderma capillare in Iran Mycoflore

Introduction: Trichoderma is monophyletic (16), with teleomorphs in the genus Hypocrea. Some cryptic Trichoderma species are hidden within morphological species complexes and can only be elucidated by in-depth molecular studies. The genealogical concordance phylogenetic species recognition (GCPSR) using several non-linked genes are needed to give accurate identification of Trichoderma spp. (6). Although the ITS region has been successfully used for species delimitation of Trichoderma and Hypocrea (5), but, it is not sufficient for accurate identification of some species. Translation elongation factor 1α gene (tef1α) is a reliable barcode for Fusarium (9), Trichoderma and Hypocrea (5). Here, ITS and tef1α genes were selected as candidate DNA barcodes to identify Trichoderma isolates. Material and methods: 40 Trichoderma isolates used in this study were from a fungal collection archived in the plant pathology laboratory in the Department of Plant Protection at the Shahid Chamran University of Ahvaz. Spore suspension (105/ml) prepared from single spore cultures of each Trichoderma isolates was added into flasks containing PDB medium. The flasks were shaken at 180 rpm for 10-15 days at 28ºC and the biomass was harvested by passing through sterilized filter papers. The mycelia were freeze-dried (Freeze-Dryer, Alpha 1-2LD Plus, Christ) and powdered in the mortar containing liquid nitrogen by pestle. The genomic DNA was isolated according to modified method established by Raeder and Broda (21). The universal primers (ITS1–F; 5'-TCCGTAGGTGAACCTGCGG-3' and ITS4-R; 5'-TCCTCCGCTTATTGATATGC-3') were employed for amplifying around 700bp from 18s, ITS1, 5.8s, ITS2 and 28s rDNA regions (27). The specific primers (tef1α71-f; 5'-CAAAATGGGTAAGGAGGASAAGAC-3' and tef1997-R; 5'-CAGTACCGGCRGCRATRATSAG-3') were employed for amplifying around 950bp from tef1α gene (24). PCR products were purified through ethanol-precipitation method and then sequenced using forward and reverse primers by Macrogen Company. The Sequences were edited and assembled using BioEdit v. 7.0.9.0 (10) and DNA Baser Sequence Assembeler v4 programs (2013, Heracle BioSoft, www.DnaBaser.com), respectively. These sequences were submit-queried against the NCBI non-redundant database and related to known DNA sequences by BLASTn algorithm to assign putative identity. They also were subjected to the TrichO Key (5) and TrichoBLAST (15) for more characterization. The phylogenetic tree was constructed through maximum likelihood analysis based on tef1α sequence under K2+G model. The tree was rooted to close species of N. macroconidialis. Result and Discussion: Approximately 550 and 850 bases of the ITS and tef1α regions were sequenced from the isolates studied and then deposited in the GenBank (Table 2). The annotation of indexed sequences showed which multiple insertion-type frame shifts have interestingly occurred into the reading frame of tef1α gene belonging to isolate of T. capillare Isf-7 (Fig. 3). To identify isolates of Trichoderma, ITS and tef1α sequences were subjected to the TrichO Key (5), TrichoBLAST (15) and BLASTn Search. The analysis of ITS and tef1α sequences (Table 2, Fig. 2), in combination with morphology (Table 1), showed which the isolates place in seven species as follow: T. harzianum Rifai, T. virens (J.H. Mill., Giddens & A.A. Foster) Arx, T. pleuroticola Yu & Park, T. asperellum Samuels, Lieckf & Nirenberg, T. koningiopsis Oudem., T. brevicompactum Kraus, Kubicek & Gams and T. capillare Samuels & Kubicek. In BLASTn search, ITS and tef1α regions separately provided unambiguous identification for isolates of T. virens, T. koningiopsis and T. brevicompactum while ITS region provided ambiguous identification for Isolates of Trichoderma harzianum, T. capillare, T. pleuroticola and T. asperellum. Here, tef1α region could provide more accurate identification as good DNA barcoding (Table 2). The isolates showed the sequence identity ranging from 96 to 100% for tef1α locus and 88 to 99% for ITS locus. Different identities related to ITS and tef1α genes indicated that the single gene identification is not accurate, particularly for Trichoderma species, if the identification is based on ITS regions (4). In phylogenetic tree (Fig. 2), the isolates surveyed generated strongly supported clades for each species, distinct from other species. Among the species identified, T. capillare is the first report for Iran mycoflora. This species was firstly described by Samuels et al. (22) and phylogenetically delimited from other species of Longibrachiatum section. Conclusions: Here, of seven species of Trichoderma identified, the species of T. capillare is newly reported in Iran. Our studies demonstrate ultimately that, despite ITS region, tef1α gene is quite reliable in identification and phylogeny of Trichoderma species. Material and methods: 40 Trichoderma isolates used in this study were from a fungal collection archived in the plant pathology laboratory in the Department of Plant Protection at the Shahid Chamran University of Ahvaz. Spore suspension (105/ml) prepared from single spore cultures of each Trichoderma isolates was added into flasks containing PDB medium. The mycelia were harvested from the growth medium by washing biomass with sterilized distilled water on filter papers. Mycelial biomasses were freeze-dried and then powdered into mortar containing liquid nitrogen by pestle. The genomic DNA was isolated according to modified method established by Reader and Broda (1985). The universal primers (ITS1–F; 5'-TCCGTAGGTGAACCTGCGG-3' and ITS4-R; 5'-TCCTCCGCTTATTGATATGC-3') were employed for amplifying around 700bp from 18s, ITS1, 5.8s, ITS2 and 28s rDNA regions. The specific primers (tef1α71-f; 5'-CAAAATGGGTAAGGAGGASAAGAC-3' and tef1997-R; 5'-CAGTACCGGCRGCRATRATSAG-3') were employed for amplifying around 950bp from exon1 to exon6 regions of tef1α gene containing introns 1 to 5 (Shoukouhi and Bisset, 2008). PCR products were purified through ethanol-precipitation method and then sequenced using forward and reverse primers by Macrogen Company. The Sequences were edited and assembled using BioEdit v. 7.0.9.0 (Hall 1999) and DNA Baser Sequence Assembeler v4 programs (2013, Heracle BioSoft, www.DnaBaser.com), respectively. These sequences were submit-queried against the NCBI non-redundant database and related to known DNA sequences by BLASTn algorithm to assign putative identity. They also were subjected to the TrichO Key (Druzhinina et al. 2005) and TrichoBLAST (Kopchinskiy et al. 2005) for more detection. Result and Discussion: Approximately 550 and 850 bases of the ITS and tef1α regions were sequenced from the isolates studied and then deposited in the GenBank. There was no ITS sequence of T. capillare in databases and we here indexed it and more sequence from its tef1α gene in GenBank. The annotation of indexed sequences showed which multiple insertion-type frame shifts have interestingly occurred into reading frame of tef1α gene belong to T. capillare Isf-7 isolate (Fig. 1). To identify isolates of Trichoderma, ITS and tef1α sequences were subjected to the TrichO Key (Druzhinina et al. 2005), TrichoBLAST ( Kopchinskiy et al. 2005) and BLASTn Search. The analysis and comparison of ITS and tef1α data with reference sequences in ISTHT and GenBank showed which the isolates place in seven species as follow: T. harzianum Rifai, T. virens (J.H. Mill., Giddens & A.A. Foster) Arx, T. pleuroticola Yu & Park, T. asperellum Samuels, Lieckf & Nirenberg, T. koningiopsis Oudem., T. brevicompactum Kraus, Kubicek & Gams and T. capillare Samuels & Kubicek. In BLASTn search, ITS and tef1α regions separately provided unambiguous identification for isolates of T. virens, T. koningiopsis and T. brevicompactum while ITS region provided ambiguous identification for Isolates of Trichoderma harzianum, T. capillare Samuels & Kubicek, T. pleuroticola and T. asperellum. Here, tef1α region could provide more accurate identification as good DNA barcoding (Table 2). The isolates showed the sequence identity ranging from 96 to 100% for tef1α locus and 88 to 99% for ITS locus. Different identities related to ITS and tef1α genes indicated that single gene identification is not accurate, particularly for Trichoderma species, if the identification is based on ITS regions (Druzhinina and Kubicek, 2005). Among the species identified, T. capillare is the first report for Iran mycoflora. This species was firstly described by Samuels et al. (2012) and phylogenetically associated with other species of Longibrachiatum Clade. Conclusions: Here, of seven species of Trichoderma identified, the species of T. capillare is newly reported in Iran. Our studies demonstrate ultimately that, despite ITS region, tef1α gene is quite reliable in identification and phylogeny of Trichoderma species

Morphological and Phylogenetic Characterization of Whip Smut on Commercial Sugarcane Cultivars and Assessing the Resistance to Sporisorium scitamineum

Whip smut, which is caused by Sporisorium scitamineum, is an important disease in areas where sugarcane is cultivated in Iran, particularly in the Khuzestan province. The pathogen significantly reduces sugarcane yield, and the use of resistant cultivars is the most cost-effective strategy for managing the disease. The present study characterized the S. scitamineum strains collected from five commercial sugarcane cultivars (CP69-1062, CP57-614, CP48-103, SP70-1143, and NCo310) based on their morphological and phylogenetic features. The sporidial cultures of the strains appeared in two growth forms: cottony colony and yeast-like. All strains were found to be identical based on the DNA sequences of ITS, COX3, GAPDH, and EF1α regions, and revealed that all strains were identical (100%) to the reference strain of S. scitamineum. The disease incidence of the cultivars varied from 5 to 43% during two consecutive years. Statistical analysis of the growth rates of the strains indicated significant differences. Combined analysis of variance (ANOVA) suggested that the effects of year, strain, cultivar, and the interaction effect of strain ´ cultivar were significant at a 1% probability level. Our results suggest that IRK310 was the most virulent among all cultivars, with different pathogenicity percentages, while the strain IRK70 had the lowest level of virulence among all strains. Among the tested cultivars, SP70-1143 and CP57-614 showed high resistance to smut. In this research, teliospore populations of whip smut were identified, and disease reactions of the cultivars were assayed. Screening and selecting smut-resistant cultivars can help reduce disease damage in cultivated areas and can serve as a basis for further research on plant disease management

Fungal Systematics and Evolution: FUSE 5

Thirteen new species are formally described: Cortinarius brunneocarpus from Pakistan, C. lilacinoarmillatus from India, Curvularia khuzestanica on Atriplex lentiformis from Iran, Gloeocantharellus neoechinosporus from China, Laboulbenia bernaliana on species of Apenes, Apristus, and Philophuga (Coleoptera, Carabidae) from Nicaragua and Panama, L. oioveliicola on Oiovelia inachadoi (Hemiptera,Veliidae) from Brazil, L. termiticola on Macrotermes subhyalinus (Blattodea, Termitidae) from the DR Congo, Pluteus cutefractus from Slovenia, Rhizoglomus variabile from Peru, Russula phloginea from China, Stagonosporopsis flacciduvarum on Vitis vinifera from Italy, Strobilomyces huangshanensis from China, Uroinyces klotzschianus on Rumex dentatus subsp. klotzschianus from Pakistan.The following new records are reported: Alternaria calendulae on Calendula officinalis from India; A. tenuissima on apple and quince fruits from Iran; Candelariella oleaginescens fromTurkey; Didymella americana and D. calidophila on Vitis vinifera from Italy; Lasiodiplodia theobromae causing tip blight of Dianella tasmanica variegata' from India; Marasmiellus subpruinosus from Madeira, Portugal, new for Macaronesia and Africa; Mycena albidolilacea, M. tenuispinosa, and M. xantholeuca from Russia; Neonectria neomacrospora on Madhuca longifolia from India; Nothophoma quercina on Vitis vinifera from Italy; Plagiosphaera immersa on Urtica dioica from Austria; Rinodina sicula from Turkey; Sphaerosporium lignatile from Wisconsin, USA; and Verrucaria murina from Turkey. Multi-locus analysis of ITS, LSU, rpbl,tefl sequences revealed that P immersa, commonly classified within Gnomoniaceae (Diaporthales) or as Sordariomycetes incertae sedis, belongs to Magnaporthaceae (Magnaporthales). Analysis of a six-locus Ascomycota-wide dataset including SS