Generation and molecular analyses of transgenic barley (Hordeum vulgare L.) in response to relevant pathogens

In plant-pathogen interaction, both host and pathogen have evolved very sophisticated strategies to survive. Plants need to resist infection and pathogens require colonizing their hosts to attain nutrients for reproduction. Plants have evolved several mechanisms to resist pathogen invasion that consists of several defence layers. Meanwhile, nonexpressor of pathogenesis-related genes 1 (NPR1) plays in the model plant Arabidopsis an essential role in systemic resistance against pathogen infection. Previous reports and recent findings have indicated a similar function for it’s homologous in rice. The significant of NPR1 in disease resistance have been proven by transient transformation of the Arabidopsis gen AtNPR1 in barley (Hordeum vulgare)-powdery mildew (Blumeria graminis f.sp. hordei, Bgh) interaction. In this respect, silencing of the NPR1 homologous gen HvNH1 using RNA interference method led to suppress NH1 transcript up to 3.7-fold at 12 hours after inoculation (hai) in challenging to Bgh. As well, in transgenic lines, the expression levels of pathogenesis-related proteins (PR-1b, PR-2 and PR-5) attenuated between 50-100% in compare to WT barley at 24 hai. The rate of susceptibility to Bgh in NH1-silenced lines increased about 29-33%. In contrary, wheat expressing AtNPR1 showed 31% increased resistance to Blumeria graminis f.sp. tritici. Histochemical observation showed suppression of hypersensitive response (HR) and cell wall apposition (CWA) by 28 and 22% in epidermal cells, respectively in NH1-silenced lines, resulted in increased fungal penetration rate. Application of systemic acquired resistance inducer, benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) induced up to 48% resistance against Bgh in WT plants, whereas this rate in transgenic plants was only 5.5%. Therefore, BTH failed in breaking down the susceptibility to Bgh in NH1-silenced line. Consistently, MLO (Mildew resistance Locus O) as a negative cell death regulator was induced up to 63-64% more in NH1- silenced lines than in WT plants at 12 and 36 hai, respectively. However, the expression level of Bax inhibitor 1 as a cell death suppressor was not affected in NH1-silenced plant. On the other hand, NH1-silenced barley had shown no difference in susceptibility to necrotrophic pathogen Fusarium graminearum, the causal agent of barley root rot. Similarly in response to leaf spot fungus, Bipolaris sorokiniana, the rate of spore penetration associated with necrotic lesions was not significantly changed in transgenic lines in compared with WT plants at 60 hai. The described phenotypes in NH1-silenced barley are inheritable and stable in different generations. In conclusion, silencing of NH1 conferred susceptibility to Bgh but not to necrotrophic and hemibiotrophic fungi. The probable mechanisms behind Bgh susceptibility in NH1-silenced plants are MLO induction as well as suppression of PR proteins.Für Pflanze-Pathogen-Interaktionen haben beide Wechselwirkungspartner ausgeklügelte Überlebensstrategien entwickelt. Pflanzen müssen sich einer Infektion erwehren, wohingegen Pathogene ihre Wirtspflanzen erfolgreich befallen müssen um an die lebensnotwendigen Nährstoffen zu gelangen. Pflanzen haben diverse Schutzmechanismen gegen einen Pathogenbefall entwickelt. Für die systemische Resistenz gegen den Pathogenbefall spielt das Gen non-expressor of pathogenesisrelated genes 1 (NPR1) in der Modellpflanze Arabidopsis eine essenzielle Rolle. Frühere und neuste Forschungsergebnisse haben eine ähnliche Funktion für dessen Homolog in Reis gezeigt. Die Bedeutung von NPR1 für die Krankheitsresistenz konnte mit Hilfe einer transienten Transformation des Arabidopsis Gens AtNPR1 auch in der Gerste (Hordeum vulgare) - Echter Mehltau (Blumeria graminis f.sp. hordei, Bgh) Interaktion gezeigt werden. Übereinstimmend konnte in stabil transgenen Gerstenpflanzen durch eine RNA-Interferenz vermittelte Suppression des NPR1-homologen HvNH1 Gens 12 Stunden nach der Inokulation (hai) mit Bgh eine 3.7-fache Unterdrückung der NH1 Transkripte gezeigt werden. Ebenfalls konnte nach einer 24-stündigen Inokulation gezeigt werden, dass sich das Expressionsniveau der pathogenesis-related Proteine (PR-1b, PR2 und PR-5) in den transgenen Linien zwischen 50-100% im Vergleich zu der Wildtyp- (WT) Gerste bewegte. In den NH1- unterdrückten Linien konnte eine 29-33%ige Anfälligkeitszunahme gegen Bgh festgestellt werden. Im Gegensatz dazu zeigte AtNPR1 exprimierender Weizen eine um 31% erhöhte Resistenz gegen Blumeria graminis f.sp. tritici. Histochemische Beobachtungen an den NH1-unterdrückten Linien wiesen eine Suppression der hypersensitiven Reaktion (HR) und von Zellwand Appositionen (CWA) in der Höhe von 28 und 22% in den epidermalen Zellen auf, die in einer erhöhten pilzlichen Penetration resultierten. Durch die Anwendung eines Induktors für die systemisch erworbene Resistenz, benzo-(1,2,3)-thiadiazole-7-carbothioic Säure S-Methylester (BTH), wurde in den WT Pflanzen eine 48%ige Resistenzerhöhung gegen Bgh erzielt, wobei diese Erhöhung in den transgenen Pflanzen nur 5.5% betrag. Demzufolge konnte BTH in den NH1-unterdrückten Linien nur eine geringe Anfälligkeitsminderung gegen Bgh hervorrufen. Entsprechend konnte gezeigt werden, dass die Expression des Gens MLO (Mehltauresistenz Locus O) als ein negativer Zelltodregulator bis zu 63-64% höher nach 12 und 24 hai in den NH1-unterdrückten Linien induziert war als in den WT Pflanzen. Jedoch war in den NH1-unterdrückten Pflanzen das Expressionsniveau von Bax Inhibitor 1 als Zelltodsuppressor nicht beeinflusst. Auf der anderen Seite zeigten die NH1-unterdrückten Gerstenpflanzen keinen Unterschied in der Anfälligkeit gegen das nekrotrophe Pathogen Fusarium graminearum, das als kausaler Erreger der Wurzelfäule bei Gerste bekannt ist. Ähnliche Ergebnisse gab es mit dem Erreger der Braunfleckigkeit, Bipolaris sorokiniana. Hierbei gab es zum Zeitpunkt 60 hai in der mit nekrotischen Lessionen verbundenen Sporenpenetrationsrate keinen signifikanten Unterschied zwischen den transgenen und WT Pflanzen. Die beschriebenen Phänotypen von der NH1-unterdrückten Gerste sind vererbbar und stabil über mehrere Generationen. Zusammengefasst kann gesagt werden, dass die Unterdrückung des NH1 Gens Anfälligkeit gegen Bgh verursacht aber nicht gegen nekrotrophe und heminekrotrophe Pilze. Die wahrscheinlichen Mechanismen der beobachteten Bgh-Anfälligkeit in den NH1-unterdrückten Pflanzen beruhen auf der MLO Induzierung wie auch auf der Unterdrückung der PR-Proteine

First report of fruit spot of pomegranate caused by Colletotrichum Gloeosporioides in Iran

Pomegranate (Punica granatum) is one of the most important commercial fruit crop in eastern Mazandaran (Iran, 35°47’N, 50°34’E). During spring 2013, distinct dark brown spots were observed on pomegranate fruits, from which a fungus was isolated on standard potato dextrose agar (PDA) amended with streptomycin (0.05% w/v). The mycelium was white- grey turning olive green over time, and produced oval to cylindrical, hyaline, unicellular, aseptate conidia measuring 5-13×1.5-4 μm. Based on these morphological characters the mycete was tentatively identified as Colletotrichum gloeosporioides. The fungal internal transcribed spacer (ITS) region of r-DNA was then amplified using the primers ITS5/ITS4 sequenced locally and deposited under GeneBank accession No. KJ769129. A sequence similarity search performed using BLAST (Altschul et al. 1990) algorithm available via GenBank confirmed the identification as C. gloeosporioides. Pathogenicity tests were carried out by placing agar-discs from a six-day-old culture of the fungus onto five artificially injured pomegranate fruits, which were placed inside sterile plastic bags. Controls consisted of non- inoculated fruits. Symptoms were reproduced after six days only on inoculated fruits and the pathogen was subsequently re-isolated, fulfilling Koch’s postulates. To our knowledge, this is the first report of. C. gloeosporioides in pomegranate fruits in Iran.http://www.sipav.org/main/jpp/index.php/jppam2016Microbiology and Plant Patholog

Essential oil composition of sweet basil (Ocimum basilicum L.) in symbiotic relationship with Piriformospora indica and paclobutrazol application under salt stress

Essential oil content and oil composition of paclobutrazol treated sweet basil (Ocimum basilicum L.) plant inoculated with Piriformospora indica under salt stress were investigated by GC-MS. The results show a slight increase in essential oil content when basil plants subjected to moderate salinity stress (3 dS m−1 of NaCl). It decreased signifiicantly with increasing salinity level to 9 dS m−1. The findings revealed that leaf area, above ground and leaf dry weights, essential oil content and yield were significantly affected by P. indica inoculation, however paclobutrazol application significantly influenced essential oil yield but not content. Fungal symbiosis as well as paclobutrazol application ameliorated the negative effects of salinity on dry matter and essential oil yield. The main constituents found in the volatile oil of O. basilicum in control treatment were Geranial (26.03%), Neral (24.88%) and Estragole (24.78%). The compounds concentrations showed some differences in P. indica and paclobutrazol treatments. The results demonstrate that micorrhiza-like fungi concomitantly increase essential oil production and biomass in sweet basil, a medicinal herb rich in commercially valuable essential oils

Molecular data confirm the mitosporic state of Hyphodermella rosae (Phanerochaetaceae) as the pathogen of rosaceous fruits in northern Iran

Hyphodermella is a genus of corticioid white rot fungi from the Phanerochaetaceae. Molecular data (partial SSU, LSU and complete ITS nrDNA) confirm the mitosporic state of Hyphodermella rosae as the causal agent of dry fruit rot of plum (Prunus domestica) and peach (Prunus persica) from Mazandaran, Iran. The asexual state of H. rosae is characterized by the presence of unicellular, spherical mitospores, which are terminal or intercalary in position. Both basidia and mitospores were observed in the matured cultures of H. rosae, although we did not observe any basidiospores. The phylogeny of Phanerochaetaceae confirms Hyphodermella as a monophyletic lineage within the family and sister group to Phanerochaete with considerable bootstrap support. Both the isolates of mitosporic H. rosae nest within a clade, which includes its sexual counterpart. Hence, we conclude that the lifecycle of H. rosae includes at least two reproductive states, i.e. sexual and asexual. Mitosporic H. rosae is capable of infecting plants and produce similar disease symptoms as its sexual state.http://www.sydowia.at/syd62-1/syd62-1.htm2016-12-30am2016Forestry and Agricultural Biotechnology Institute (FABI

Insect peptide metchnikowin confers on barley a selective capacity for resistance to fungal ascomycetes pathogens

The potential of metchnikowin, a 26-amino acid residue proline-rich antimicrobial peptide synthesized in the fat body of Drosophila melanogaster was explored to engineer disease resistance in barley against devastating fungal plant pathogens. The synthetic peptide caused strong in vitro growth inhibition (IC50 value ∼1 μM) of the pathogenic fungus Fusarium graminearum. Transgenic barley expressing the metchnikowin gene in its 52-amino acid pre-pro-peptide form under the control of the inducible mannopine synthase (mas) gene promoter from the Ti plasmid of Agrobacterium tumefaciens displayed enhanced resistance to powdery mildew as well as Fusarium head blight and root rot. In response to these pathogens, metchnikowin accumulated in plant apoplastic space, specifying that the insect signal peptide is functional in monocotyledons. In vitro and in vivo tests revealed that the peptide is markedly effective against fungal pathogens of the phylum Ascomycota but, clearly, less active against Basidiomycota fungi. Importantly, germination of the mutualistic basidiomycete mycorrhizal fungus Piriformospora indica was affected only at concentrations beyond 50 μM. These results suggest that antifungal peptides from insects are a valuable source for crop plant improvements and their differential activities toward different phyla of fungi denote a capacity for insect peptides to be used as selective measures on specific plant diseases

The ‘Green Revolution’ dwarfing genes play a role in disease resistance in Triticum aestivum and Hordeum vulgare

The Green Revolution dwarfing genes, Rht-B1b and Rht-D1b, encode mutant forms of DELLA proteins and are present in most modern wheat varieties. DELLA proteins have been implicated in the response to biotic stress in the model plant, Arabidopsis thaliana. Using defined wheat Rht near-isogenic lines and barley Sln1 gain of function (GoF) and loss of function (LoF) lines, the role of DELLA in response to biotic stress was investigated in pathosystems representing contrasting trophic styles (biotrophic, hemibiotrophic, and necrotrophic). GoF mutant alleles in wheat and barley confer a resistance trade-off with increased susceptibility to biotrophic pathogens and increased resistance to necrotrophic pathogens whilst the converse was conferred by a LoF mutant allele. The polyploid nature of the wheat genome buffered the effect of single Rht GoF mutations relative to barley (diploid), particularly in respect of increased susceptibility to biotrophic pathogens. A role for DELLA in controlling cell death responses is proposed. Similar to Arabidopsis, a resistance trade-off to pathogens with contrasting pathogenic lifestyles has been identified in monocotyledonous cereal species. Appreciation of the pleiotropic role of DELLA in biotic stress responses in cereals has implications for plant breeding

Induction of Wheat Resistance to STB by the Endophytic Fungus serendipita Indica and pseudomonas Protegens.

Septoria tritici blotch (STB) caused by fungus Zymoseptoria tritici, is one of the important wheat (Triticum aestivum L.) diseases difficult to control because of the lack of wheat resistant cultivars. The use of biological control agents is one possible way for triggering host plant resistance to biotic and abiotic stresses. In this study, we examined the ability of Serendipita indica and Pseudomonas protegens CHA0-mCherry in inducing the local wheat cultivar Tajan resistance to STB. The interaction between biological control agents and the roots of wheat was evaluated. The experiment was conducted in a completely randomized design by three replicates. Spore suspension was supplied at concentrations of 10 <sup>7</sup> and 10 <sup>9</sup> for S. indica and bacteria isolate (CHA0-mCherry) respectively. Five treatments were applied including S. indica, CHA0-mCherry, S. indica and CHA0-mCherry co-inoculation, positive and negative control. Twenty-one days after inoculation, the interaction between biological agents and plant roots were evaluated through morphological traits and qPCR. The plant resistance, disease severity, and the correlation between resistance and disease severity were assessed. Pycnidial variation and agronomic traits were also evaluated. Twenty-one days after inoculation, both biological agents clearly colonized all treated roots of all treatments except in control plants as demonstrated by qPCR analysis. Chlamydospores were observed in the S. indica-treated hosts with the CHA0-mCherry colonizing assessment showing 5×109 CFU g-1 in the root. The asexual phase of the fungal pathogen, pycnidial diameter, was reduced in S. indica treated plants more considerably than in the other treatments. There was a positive correlation between resistance and disease severity mean when calculated by Pearson's correlation. There was a significant difference between the root length, fresh, and dry weight of root. Spore density was inversely correlated to resistance and disease severity, when compared with control, with CHA0-mCherry being the most effective in reducing the spore density. S. indica was the most effective in promoting root growth and stem biomass, when compared with control. Serendipita indica and Pseudomonas protegens CHA0-mCherry colonies showed a potential biological control activity and efficiently enhanced the plant resistance to Z. tritici in the treated wheat roots. The microbial biological control agents are very practical in crop protection against plant disease and can be very useful in sustainable agriculture. PLSN: percentage of leave surface necrosis, DPI: day past inoculation, PLACL: percentage of leaf area covered by lesions, PPMLA: pycnidia per millimeter in leaf area

Expression of the pathogenesis related proteins, NH-1, PAL, and lipoxygenase in the iranian Tarom and Khazar rice cultivars, in reaction to Rhizoctonia solani – the causal agent of rice sheath blight

Pathogenesis related (PR) genes of rice are among the most important defense genes in the interaction of rice with pathogens. In this study, the role of NH-1, several PR genes, phenylalanine ammonia-lyase (PAL), and lipoxygenase in the defense responses of rice against Rhizoctonia solani, the causal agent of rice sheath blight disease, was evaluated. The Tarom and Khazar cultivars (cvs), as resistant and susceptible genotypes, respectively, were used. The expression rate of defense genes in two-week-old seedlings inoculated with a virulent isolate of R. solani AG-I-1 A was investigated. The lesions in the Tarom cv were less than half the size of those on the Khazar cv. The expression scripts of the genes were calculated by quantative Real-Time PCR (RT-PCR). Results showed that the expression rate of all genes in the resistant cultivar was higher than that of the susceptible genotype, post inoculation. Analysis of data by the t-Student test also indicated significant differences in the expression level of the genes between Khazar and Tarom. The results of this study suggest that the investigated genes are involved in the resistance responses of rice against the sheath blight agent. For the first time, the induction of PR-5, PR-9, PR-10, PR-12, PR-13, and NH-1 was observed in this study in the resistant and susceptible Iranian cultivars of rice following attacks by R. solani

Isolation, Molecular Identification, and Mycotoxin Production of Aspergillus Species Isolated from the Rhizosphere of Sugarcane in the South of Iran

Knowledge of the genetic diversity detected among fungal species belonging to the genus Aspergillus is of key importance for explaining their important ecological role in the environment and agriculture. The current study aimed to identify Aspergillus species occurring in the rhizosphere of sugarcane in the South of Iran, and to investigate their mycotoxin profiles. One-hundred and twenty-five Aspergillus strains were isolated from the soil of eight major sugarcane-producing sites, and were molecularly identified using sequences of partial -tubulin (benA) and partial calmodulin (CaM) genes. Our molecular and phylogenetic results showed that around 70% of strains belonged to the Aspergillus section Nigri, and around 25% of species belonged to the Aspergillus section Terrei. Species belonging to both sections are able to produce different mycotoxins. The production of mycotoxins was measured for each species, according to their known mycotoxin profile: patulin (PAT) and sterigmatocystin (STG) for Aspergillus terreus; ochratoxin A (OTA) and fumonisins for Aspergillus welwitschiae; and OTA alone for Aspergillus tubingensis. The data showed that the production of OTA was detected in only 4 out of 10 strains of A. welwitschiae, while none of the A. tubingensis strains analyzed produced the mycotoxin. Fumonisins were produced by 8 out of 10 strains of A. welwitschiae. Finally, none of the 23 strains of A. terreus produced STG, while 13 of them produced PAT. The occurrence of such mycotoxigenic plant pathogens among the fungal community occurring in soil of sugarcane fields may represent a significant source of inoculum for the possible colonization of sugarcane plants, since the early stages of plant growth, due to the mycotoxin production capability, could have worrisome implications in terms of both the safety and loss of products at harvest