Spectral pattern study of citrus black rot caused by Alternaria alternata and selecting optimal wavelengths for decay detection.

Fungal decay is one of the most common diseases that affect postharvest operations and sales of citrus. Sometimes, fungal disease develops and spreads inside the fruit and in the advanced stages of the disease, it appears apparent, so the use of efficient and reliable methods for early detection of the disease is very important. In this study, early detection of citrus black rot disease caused by Alternaria genus fungus was examined using spectroscopy. Jaffa oranges were inoculated with Alternaria alternata. The samples were inspected by spectroscopy (200-1100 nm) in the 1st, 2nd, and 3rd weeks after inoculation. The classification of healthy and infected samples and selection of most important wavelengths were conducted by soft independent modeling of class analogy (SIMCA). The most important wavelengths in the detection of healthy and infected samples of the 1st week were 507, 933, 937, and 950 nm with a classification accuracy of 60%. The most important wavelengths of the 2nd week were 522 and 787 nm with a classification accuracy of 60%. Also, wavelengths of 546, 660, 691, and 839 were found to be effective in the 3rd week with a classification accuracy of 100%.

Differentiation of an Iranian resistance chickpea line to Ascochyta blight from a susceptible line using a functional SNP.

Identification of resistant sources to Ascochyta blight (AB) has been considered as a main purpose in most chickpea breeding programs. Achievements to molecular markers related to resistance to Ascochyta rabiei allows selection programs to be developed more accurately and efficiently. The aim of this study was to investigate the applicability of a functional SNP in differentiating Iranian resistant cultivars to be used in selection programs. Amplification of SNP-containing fragment with specific primer pair and its sequencing resulted in tracking and determining the allelic pattern of SNP18, SNP18-2147, SNP18-2491 and SNP18-2554 loci belong to GSH118 gene in ILC263 (sensitive) and MCC133 (resistant) chickpea lines. Mutations in SNP18 and SNP18-2147 occur at the protein level at positions 499 and 554. Bioinformatics studies have shown that the GSH118 gene is a Lucien-rich repeat receptor kinases (LRR-RKs) and encodes a membrane protein which can be involved in recognizing microorganisms and initiating immune signaling pathways in plants. Additional studies to determine the function of this gene and its interaction with other proteins can be effective in gaining more knowledge about the molecular basis of resistance against AB.

Transient expression analysis of synthetic promoters containing F and D cis-acting elements in response to Ascochyta rabiei and two plant defense hormones.

Introduction of a foreign gene coding for a pathogen resistant protein into the target plant and constitutive expression of Resistance (R) proteins may confer high level of resistance. However, genetic engineering could lead to reprogramming of molecular mechanisms that manage physiological behavior, which in turn could lead to undesired results. Therefore, using a pathogen-inducible synthetic promoter approach, response to pathogens could be more specific. Ascochyta rabiei is a destructive fungal pathogen in chickpea production. In this study, we analyzed the expression pattern of three synthetic promoters in response to pathogen and two defense hormones. We have tested three synthetic pathogen-inducible promoters designated as (1) synthetic promoter-D box-D box (SP-DD), (2) synthetic promoter-F element-F element (SP-FF) and (3) synthetic promoter-F element-F element-D box-D box (SP-FFDD) via Agrobacterium transient expression assay. The cis-acting element designated as 'D' is a 31 base pair sequence from the promoter of parsley pathogenesis-related gene 2 (PR2 gene) and the cis-acting element designated as 'F' is a 39 base pairs sequence from the promoter of Arabidopsis AtCMPG1 gene. We used mycelial extracts from two pathotypes of A. rabiei as elicitor to define the responsiveness of the promoters against pathogen. Plant phytohormones including salicylic acid and methyl jasmonate were also used to study the promoter sensitivity in plant signaling pathways. Our results showed that the SP-FF promoter was highly inducible to A. rabiei and methyl jasmonate as well, while the SP-DD promoter was more sensitive to salicylic acid. The SP-FFDD promoter was equally responsive to both pathotypes of A. rabiei which is probably due to the complex nature of box D cis-acting element.

Study on Antiviral Activity of Two Recombinant Antimicrobial Peptides Against Tobacco Mosaic Virus.

Antimicrobial peptides (AMPs) are generally small peptides with less than 50 amino acid residues, which have been considered as the first line of defense system in plants and animals. These small cationic peptides belong to a family of antimicrobials that are multifunctional effectors of innate immunity. The direct antimicrobial activity of AMPs against different bacteria, viruses, fungi, and parasites has been confirmed in different studies. In this study, the antiviral activity of two recombinant AMPs named thanatin and lactoferricin+lactoferrampin was evaluated against Tobacco mosaic virus (TMV) using half-leaf and leaf disk methods under in vivo and in vitro condition, respectively. The obtained result indicated that both recombinant AMPs have shown an antiviral activity against TMV. Compared to the chimeric lactoferricin+lactoferrampin, recombinant thanatin showed a higher rate of antiviral activity against TMV. Three types of effects, including protective, curative, and inactivation, were evaluated during an antiviral activity test. In the present study, the antiviral activity of two recombinant AMPs is represented for the first time: thanatin and chimeric lactoferricin+lactoferrampin against TMV as a viral plant pathogen.

Antifungal activity of recombinant thanatin in comparison with two plant extracts and a chemical mixture to control fungal plant pathogens.

The most common method for controlling plant diseases is the application of chemical pesticides and sometimes use of resistant cultivars. Due to the effects of chemical pesticides on human and environmental health, mutation in pathogens and resistance to various toxins besides the challenges with resistant cultivar production, the constant use of these methods are not recommended any longer. Thus, use of biological control agents along with the natural ingredient extracted from plants and application of peptide with antimicrobial activity, have been the focus of many researchers. In the present study, the antifungal activity of two plant extracts named Turmeric and Persian lilac in comparison with a chemical mixture and recombinant thanatin were evaluated against five following fungal plant pathogens; Geotrichum candidum, Botrytis cinerea, Rhizoctonia solani, Alternaria tenuissima and Gibberella fujikuroi. The results showed that, all treatments have antifungal activity against tested fungi. Both plant extracts were shown an acceptable antifungal activity against tested fungi but their inhibition effects was not comparable with chemical mixture. Turmeric showed a higher rate of mycelial inhibition than Persian lilac. Amongst all treatment, thanatin showed a great antifungal activity by its application at µg level under both in vitro and in vivo condition. Considering to the compatibility of thanatin with human health and environmental safety we could imagine a clear perspective for the application of this recombinant peptide in sustainable agriculture.

Heterologous expression of a broad-spectrum chimeric antimicrobial peptide in Lactococcus lactis: Its safety and molecular modeling evaluation.

Over the last decade, global increase in antibiotic consumption is a major concern in the word. Antimicrobial peptides (AMPs) known as potential alternative and were considered as a safe antimicrobial agent. However, current approaches for production and purification of AMPs are costly and time-consuming. Here we show that heterologous expression of a chimeric peptide was successfully developed in Lactococcus lactis as a safe and cost-effective recombinant protein expression platform. Minimum inhibitory concentrations (MICs) of His-tag purified peptide was determined against a broad spectrum of human pathogenic bacteria consistence of Gram-positive, Gram-negative and resistance strains in deferent range from 7.24 ±â€¯0.4 to 156.24 ±â€¯3.0 µg/mL. Furthermore, our results showed that the peptide was not toxic to HEK and HeLa cells and even at concentrations as high as 250 µg/mL exhibited minimal hemolysis against RBCs. Additional characteristics such as thermal, protease and 50% human plasma stability were determined for cLFchimera. Molecular modeling analysis demonstrated that fusion of His-tag to the C-terminal of chimeric peptide increased peptide stability during 10 ns simulation in water. Overall, the chimeric peptide has a considerable antibacterial activity with low hemolysis, low or none in toxicity and good temperature resistance and also high stability in serum. We anticipate the established expression system could be developed and used more effectively in probiotic strains in future studies.

Expression and Purification of the Main Component Contained in Camel Milk and Its Antimicrobial Activities Against Bacterial Plant Pathogens.

Lactoferrin is the most dominant protein in milk after casein. This protein plays a crucial role in many biological processes including the regulation of iron metabolism, induction and modulation of the immune system, the primary defense against microorganisms, inhibiting lipid peroxidation and presenting antimicrobial activity against various pathogens such as parasites, fungi, bacteria, and viruses. The major antimicrobial effect of lactoferrin is related to its N-terminal tail where different peptides for instance lactoferricin and lactoferrampin which are important for their antimicrobial abilities are present. The growth rate of bacterial cells in camel milk is lower than that of the cow milk due to having more antimicrobial compounds. In this study, we have fused a codon-optimized partial camel lactoferrcin and lactoferrampin DNA sequences in order to construct a fused peptide via a lysine. This chimeric 42-mer peptide consists of complete and partial amino acid sequence of camel lactoferrampin and lactoferricin, respectively. Human embryonic kidney 293 (HEK-293) cells were used for synthesizing this recombinant peptide. Finally, the antibacterial activities of this constructed peptide were investigated under in vitro condition. The result showed that, all construction, cloning and expression processes were successfully performed in HEK-293. One His-tag tail was added to the chimera in order to optimize the isolation and purification processes and also reduce the cost of production. Additionally, His-tag retained the antimicrobial activity of the chimera. The antimicrobial tests showed that the growth rate in the majority of bacterial plant pathogens, including gram negative and positive bacteria, was inhibited by recombinant chimera as the level of MIC values were evaluated between 0.39 and 25.07 µg/ml for different bacterial isolates.

Detection and classification of citrus green mold caused by Penicillium digitatum using multispectral imaging.

BACKGROUND: Fungal decay is a prevalent condition that mainly occurs during transportation of products to consumers (from harvest to consumption) and adversely affects postharvest operations and sales of citrus fruit. There are a variety of methods to control pathogenic fungi, including UV-assisted removal of fruit with suspected infection before storage, which is a time-consuming task and associated with human health risks. Therefore it is essential to adopt efficient and dependable alternatives for early decay detection. In this study, detection of orange decay caused by Penicillium genus fungi was examined using spectral imaging, a novel automated inspection technique for agricultural products. RESULTS: The reflectance parameter (including mean reflectance) and reflectance distribution parameters (including standard deviation and skewness) of surfaces were extracted from decayed and rotten regions of infected samples and healthy regions of non-infected samples. The classification accuracy of rotten, decayed and healthy regions at 4 and 5 days after fungal inoculation was 98.6 and 100% respectively using the mean and skewness of 500 nm, 800 nm, 900 nm and modified normalized difference vegetation index (MNDVI) spectra. CONCLUSION: Comparison of results between healthy and infected samples showed that early real-time detection of Penicillium digitatum using multispectral imaging was possible within the near-infrared (NIR) range. © 2018 Society of Chemical Industry.

Application of surface modified nano ferrite nickel in catalytic reaction (epoxidation of alkenes) and investigation on its antibacterial and antifungal activities.

Newly, magnetic nanoparticles have extensively been used as alternative catalyst supports, in the view of their high surface area which results in high catalyst loading capacity, high dispersion, low toxicity, environmental preservation, distinguished stability, and suitable catalyst reusing. In the present study, the magnetite nanoparticles, NiFe2O4@Ag and NiFe2O4@Mo, were synthesized and characterized. The antimicrobial activities and catalytic properties of synthesized nanoparticles were tested afterwards. For synthetizing the nanoparticle NiFe2O4@Ag, silver ions were loaded onto the surface of the modified NiFe2O4 and reduced to silver crystal by adding NaBH4. The antibacterial effects of NiFe2O4@Ag were examined against two species of soil and plant related bacteria named Bacillus subtilis (gram positive) and Pseudomonas syringae (gram negative), respectively. The antifungal activity of this nanoparticle was evaluated against two species of plant pathogenic fungi called Alternaria solani and Fusarium oxysporum. Biological results indicated that the synthesized material has shown an excellent antibacterial and antifungal activity against all examined bacteria and fungi so that, their growth were completely inhibited 24h after treatment with NiFe2O4@Ag. For the synthesis of a heterogeneous catalyst NiFe2O4@Mo, complex Mo(CO)6 was loaded onto the surface of the modified NiFe2O4 nanoparticle. This catalyst was found as an efficient catalyst for epoxidation of cis-cyclooctene and a wide variety of alkenes, including aromatic and aliphatic terminal ones using tert-butyl hydroperoxide as oxidant. This new heterogenized catalyst could easily be recovered by using a magnetic separator and reused four consecutive and loss only 13% of its catalytic activity.

An N-acetyl-beta-D-glucosaminidase gene, cr-nag1, from the biocontrol agent Clonostachys rosea is up-regulated in antagonistic interactions with Fusarium culmorum.

Clonostachys rosea is a widely distributed fungus that often acts as a parasite on other soil fungi. This fungus has also been reported as a potential parasite against nematodes and insects. The antagonistic activity is thought to be correlated with the secretion of cell wall-degrading enzymes, including chitinases. In this work, we identified and characterized an N-acetyl-beta-D-glucosaminidase-encoding gene, cr-nag1, belonging to glycosyl hydrolase family 20, from the C. rosea strain IK726 using a degenerated primer strategy designed from conserved motifs. The complete gene, including its promoter region, was obtained by genomic walking. Southern analysis showed that cr-nag1 is present as a single copy gene in C. rosea. Phylogenetically, cr-nag1 showed the highest similarity to N-acetyl-beta-d-glucosaminidase genes from other mycoparasitic fungi. Enzymatic assays and RT-PCR showed that the NAGase activity of C. rosea is specifically repressed in medium containing a high glucose content and is expressed in media containing chitin or Fusarium culmorum cell walls as sole carbon sources. Macroscopic and microscopic observations indicated that the mycelial growth of F. culmorum and Pythium ultimum were inhibited during interactions with C. rosea. High expression of cr-nag1 was found in interactions between C. rosea and F. culmorum, whereas the expression of cr-nag1 in interactions between C. rosea and P. ultimum was similar to the control. This indicates that although C. rosea secretes chitin-hydrolysing agents in order to target the cell wall of F. culmorum, it seems to use another strategy for controlling the development of the oomycete P. ultimum.

Real-time RT-PCR expression analysis of chitinase and endoglucanase genes in the three-way interaction between the biocontrol strain Clonostachys rosea IK726, Botrytis cinerea and strawberry.

Clonostachys rosea is a well-known biocontrol agent against Botrytis cinerea, the causal agent of gray mold in strawberry. The activity of cell wall-degrading enzymes might play a significant role for successful biocontrol by C. rosea. The expression pattern of four chitinases, and two endoglucanase genes from C. rosea strain IK726 was analyzed using real-time RT-PCR in vitro and in strawberry leaves during interaction with B. cinerea. Specific primers were designed for beta-tubulin genes from C. rosea and B. cinerea, respectively, and a gene encoding a DNA-binding protein (DBP) from strawberry, allowing in situ activity assessment of each fungus in vitro and during their interaction on strawberry leaves. Growth of B. cinerea was inhibited in all pathogen-antagonist interactions while the activity of IK726 was slightly increased. In all in vitro interactions, four of the six genes were upregulated while no change in expression of two endochitinases was measured. In strawberry leaves, the chitinase genes were upregulated 2-12-fold, except one of the endochitinases, whereas no change in expression of the two endoglucanases was measured. The results suggest that three out of four chitinase genes of IK726 are involved in biocontrol on leaves. This is the first example of monitoring of expression of chitinolytic genes in interactions between biocontrol agents and pathogens in plant material.

Three endochitinase-encoding genes identified in the biocontrol fungus Clonostachys rosea are differentially expressed.

Three endochitinase-encoding genes, cr-ech58, cr-ech42 and cr-ech37 were identified and characterised from the mycoparasitic C. rosea strain IK726. The endochitinase activity was specifically induced in media containing chitin or Fusarium culmorum cell walls as sole carbon sources. RT-PCR analysis showed that the three genes were differentially expressed. The expression of the cr-ech42 and cr-ech37 genes was triggered by F. culmorum cell walls and chitin whereas glucose repressed their expression. In contrast, the expression of cr-ech58 was not triggered by F. culmorum cell walls and chitin, suggesting a different role for this endochitinase. Phylogenetically, the cr-ech42 and cr-ech37 genes showed to be orthologous to endochitinase 42 and 37 kDa encoding genes from other mycoparasitic fungi, while no orthologous gene for the cr-ech58 gene was found. Three genetically modified mutants of C. rosea were made by disruption of the endochitinase genes via Agrobacterium-mediated transformation and their biocontrol activity was evaluated. While in planta bioassays showed no significant difference in biocontrol efficacy between the disruptants and the wildtype, the real time RT-PCR analysis showed that disruption of each endochitinase gene affected the activity of C. rosea during interaction with F. culmorum in liquid cultures.