Comparative Analysis of Antifungal Activity of Total Phenolics from Different Date Palm Cultivars Against Five Phyto Pathogenic Fungi

Introduction: Pathogen attacks impose natural selection on plants to evolve complex arrays of defensive strategies. Among the diverse defensive mechanisms evolved by plants to withstand pathogen attack, the ability to synthesize an arsenal of low-molecular weight volatile and non-volatile chemicals including phenolics helps them to prepare a robust defense response against pathogen entry. Systemic induction and accumulation of low molecular weight phenolics is observed in response to various diseases and thus are studied as markers for resistance to pathogens. Phenolics that exhibit anti-oxidant activity exert their inhibitory effects on pathogen colonization via protein precipitation and iron depletion. Phytochemical analysis have been proved that date palm is rich source of phenol. Very little information is available on the inherent Date palm phenolic content that has been involved as resistance factors. All the studies are focused on phenolic content from date palm fruit and its property. Here we focusing on comparative analysis of phenolics from different cultivars leaf and how it affect the different pathogenic fungi. Material and Methods: We conducted a genome mining analysis of date palm whole genome available in the NCBI site, to detect the presents of enzyme involved in the secondary metabolite pathway. Analyzed the presents of receptor protein specific for the recognition of fungal pathogen. Five date palm pathogens were isolated from the diseased date palm and surrounding soil from the date palm field located in northern region of Qatar. Leaf, shoot and root samples collected from the diseased date palm and rhizosphere soil collected from near the diseased date palm. Samples were stored at 40?C in aseptic condition until further use. Sterilized plant samples were plated in the potato dextrose agar (PDA) for the fungal isolation and the soil were plated on molten agar for fungal isolation. The plates were incubated at 250?C until single colony appeared. The isolated fungi were examined under microscope. Based on the microscopic and physical characteristics fungi were identified. The pathogenicity were determined with detached leaf inoculation analysis and in vivo pathogenicity analysis with three date palm cultivar varieties. Detached leaf inoculation analysis performed in laboratory condition and the in vivo pathogenicity conducted in green house with controlled growth condition. The date palm varieties used in this current study are Khalas, Khneezi and Barhi. All the four pathogens, Fusarium solani, Fusarium oxysporum, Rhizectonia solani Fusarium sp and Ceratocystis radicicola were used for pathogenicity analysis. Total phenolic were extracted from three date palm culvars through water extraction procedure. Extraction performed with different temperature range. Comparative analysis of antifungal property of total phenolics from different date palm cultivars such as Khalas, Khneezi and Barhi was carried out after optimizing extraction temperature. Antifungal activity is determined with disc diffusion analysis. 100 ?l of extract impregnated filter disc (10 mm in diameter) placed on the PDA plate followed by fungal disc placed on the disc. Plates were incubated at 250?C and the fungal growth monitored. Experiment repeated in triplicate along with control. Results and conclusion: The genome mining analysis of date palm result revealed 45 enzyme sequences from shikimate pathway, which is a support for the active synthesis of phenolic content in date palm. Plant phenolics synthesize via shikimate-phenylpropanoid-flavonoid pathways and include phenolic acids, flavanoids, tannins and less common stilbenes and lignins. Presents of chitin elicitor receptor kinase in date palm indicate the phytopathogenic fungal detection ability of date palm. From the isolated fungi, the date palm pathogenic fungi were screened and subcultured. Five pathogenic fungi were isolated, Fusarium solani, Fusarium oxysporum, Rhizectonia solani, Fusarium sp and Ceratocystis radicicola. Pathogenicity of all the five isolated fungi were confirmed by analyzing necrosis caused on the date palm leaf (Fig. 1). The frequency of necrotic lesion and disease susceptibility found more in Khneezi than Khalas and Barhi. Water extraction procedure conducted at 400?C for 24 hrs were accepted as standardized phenolic extract for antifungal activity. Growths of the fungi were measured after 3 day and 5 days of incubation to determine the antifungal activity of phenolic extract (table 1). Phenolic extract from the Khalas showed more antagonistic activity against Rhizectonia solani whereas phenolic extract from Barhi showed more inhibitory activity against Fusarium solani, Fusarium oxysporum and Ceratocystis radicicola. In all the experiment Khneezi showed week inhibitory activity this supports our previous susceptibility study (not published) in that Khneezi showed more susceptible to C.radicicola. This result is an evident for the disease resistant activity of date palm phenolics.qscienc

Modulation of Plant Defense System in Response to Microbial Interactions

© Copyright © 2020 Nishad, Ahmed, Rahman and Kareem. At different stages throughout their life cycle, plants often encounter several pathogenic microbes that challenge plant growth and development. The sophisticated innate plant immune system prevents the growth of harmful microbes via two interconnected defense strategies based on pathogen perception. These strategies involve microbe-associated molecular pattern-triggered immunity and microbial effector-triggered immunity. Both these immune responses induce several defense mechanisms for restricting pathogen attack to protect against pathogens and terminate their growth. Plants often develop immune memory after an exposure to pathogens, leading to systemic acquired resistance. Unlike that with harmful microbes, plants make friendly interactions with beneficial microbes for boosting their plant immune system. A spike in recent publications has further improved our understanding of the immune responses in plants as triggered by interactions with microbes. The present study reviews our current understanding of how plant–microbe interactions can activate the sophisticated plant immune system at the molecular level. We further discuss how plant-microbe interaction boost the immune system of plants by demonstrating the examples of Mycorrhizal and Rhizobial association and how these plant-microbe interactions can be exploited to engineer disease resistance and crop improvement.This study was supported by GSRA (Grant GSRA2-1-0608-14021) from the Qatar National Research Fund (a member of Qatar foundation)

Genome Mining of Secreted Effectors of Fusarium Oxysporum, Fusarium Solani and Rhizectonia Solani

Introduction: Of the various devastating diseases triggered by necrotrophic fungi, the diseases caused by following fungi Fusarium oxysporum, Fusarium solani and Rhizectonia solani are of particular economic significance in date palm (Phoenix dactylifera L.) growing countries. Undoubtedly date palm is the most important fruit tree cultivated in Arabian Peninsula. Unfortunately date palm cultivation encounter many challenges, such as fungal diseases. It has been reported that Fusarium oxysporum, Fusarium solani and Rhizectonia solani are the major causative of various diseases in Date palm. For successful colonization of host, necrotrophic pathogens secrete an arsenal of effector proteins that facilitates infection by manipulating host cell structure and function. Thus secretory proteins (SPs) are critical for both short and long range intercellular signaling during infection and colonization. The present study is conducted on three date palm pathogens, which causing diseases like bayoud disease and other necrotrophic diseases including root rot. This study focused on molecular genetics of necrotrophic pathogenicity in date palm by following an in silico approach to mine putative secreted elicitors from publically available Fusarium oxysporum, Fusarium solani and Rhizectonia solani ESTs. Methodology: Methodology involved mining of 25213 Fusarium oxysporum ESTs, 12433, Fusarium solani ESTs and 22324 Rhizectonia solani ESTs for protein sequence based features characteristic of SPs (signal peptides). All the ESTs were collected from NCBI. With using ORF predictor (http://proteomics.ysu.edu/tools/OrfPredictor.html) algorithm, found out all the six frame ORF. Predisi (http://www.predisi.de/) and SignalP (http://www.cbs.dtu.dk/services/SignalP/) algorithms was exploited to infer signal peptide within the ORF. Followed by scanning of transmembrane domain performed with TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) to discard the transmembrane sequences. 0 TM/1TM proteins were selected for further CD (conserved domain) search. Before going to the mining experiment, the pathogenicity of soil isolated (from northern part of Qatar) fungal pathogen on Date palm was confirmed with detached leaf inoculation method. All the three fungi, Fusarium oxysporum, Fusarium solani and Rhizectonia solani showed necrosis in date palm leaf after three days of infection. Result and conclusion: Out of 25213 Fusarium oxysporum ESTs 24890 have ORF. 12176 ORF found in Fusarium solani ESTs. Above 60% of all the three fungal ESTs had signal peptide. Number of sequences narrowed down by transmembrane topology search and the sequence doesn't containing transmembrane domain were selected for conserved domain identification. Subsequent to our searches with sequences lacking transmembrane domains, we could catalog candidate effector sequences based on conserved features common to characterized fungal effectors. In Fusarium oxysporum following protein found to be extracellular secretory protein, this include cellulose binding protein, which possibly involved in in host cell adhesion, qaq like polyprotein: a virulent associated protein, lysin motif: an effector protein, extracellular peptidase, cell wall hydrolase, 150 amino acid cerato platanin sequence (elicitor), alpha amylase, beta 1,3 glucanase and 165 amino acid necrosis inducing protein. These effector proteins have the ability to degrade plant derived compound, this suggest the role of the effectors in pathogenicity. In addition to these high amount of lipid targeting enzymes and glycoside hydrolase found in all the three phyto pathogenic ESTs. Presents of secretory protease detected in Fusarium solani. Peptidase like superfamily domain found in Rhizectonia solani. To date, none of these effectors have been cloned and thus we propose that the catalogued effectors will in future be useful for isolation and characterization of these identified proteins. Expression studies are already underway and we expect that the results will provide insights into mechanisms underlying host-Pythium interactions and thereby help in generating new strategies for effective date palm disease control.qscienc

Survey and identification of date palm pathogens and indigenous biocontrol agents

Fungal diseases are considered a major threat to plant growth and productivity. However, some beneficial fungi growing in the same environment protect plants from various pathogens, either by secreting antifungal metabolites or by stimulating the host immune defense mechanism. Date palms are susceptible to several fungal pathogens. Nevertheless, information on the pathogenic fungal distribution in date palm fields across different seasons is limited, especially that from Qatar. Therefore, the current study's aim was to evaluate the pathogenic and beneficial fungal diversity and distribution, including the endophytic fungi from the date palm tissues and root-associated soil fungi, during different seasons, for the identification of indigenous biocontrol agents. Our results showed that the highest number of fungal species was isolated in fall and spring, and pathogenic fungi were isolated mainly in spring. This is the first report that in Qatar, Neodeightonia phoenicum and Thielaviopsis punctulata cause date palm root rot disease, Fusarium brachygibbosum and Fusarium equiseti cause date palm wilting, and N. phoenicum causes diplodia disease in date palm offshoots. The combinations of the fungi that did not frequently occur together in date palm rhizosphere soil were investigated to identify indigenous biocontrol agents. Based on the results, we determined that Trichoderma harzianum and Trichoderma longibrachiatum are effective antagonistic fungi against T. punctulata, N. phoenicum, F. brachygibbosum, and Fusarium solani, qualifying them as potential biocontrol agents. Antagonistic activity of endophytic fungi against the pathogens was tested; except for Ulocladium chartarum, no endophytic fungi showed antagonistic activity against the tested pathogens

Antagonistic Effects of Trichoderma harzianum Isolates against Ceratocystis radicicola: pioneering a Biocontrol Strategy against Black Scorch Disease in Date Palm Trees

Date palm is an important subsistence crop in arid regions due to its ability to grow under adverse environmental conditions such as high temperature, salinity and drought. Nevertheless, ideal conditions for its growth and production are also favourable to fungal diseases such as black scorch disease caused by Ceratocystis radicicola. The aim of this study was to develop a method of biological control through the isolation, identification and examination of the effectiveness of bioagents in controlling black scorch disease. Twenty-five isolates of Trichoderma spp. were isolated from the rhizosphere of healthy date palm trees and morphological, microscopic and molecular approaches confirmed the identity of 11 isolates as Trichoderma harzianum species complex (THSC). In vivo study, application of both spore suspension and culture filtrates of T. harzianum decreased the size of necroses caused by C. radicicola. Additionally, scanning electron microscopy (SEM) showed lysis of the hyphal pathogen and phialoconidia along with scattered aleurioconidia. Results from the volatile metabolic assay and SEM suggested potential roles of cell wall degradation enzymes and volatile substances produced by T. harzianum as two collective mechanisms leading to degrade the cell wall of the pathogen and inhibit fungal growth. Altogether, results from our study demonstrated the efficacy and utility of using bioagents to control black scorch disease which could improve date palm yield.This research was made possible by a grant from the Qatar National Research Fund (QNRF) under National Priorities Research Program (NPRP-5-1002-4-010). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the QNRF. The authors would like to thank laboratory technicians in the Department of Biological and Environmental Sciences, College of Arts and Sciences, Qatar University for their technical support.Scopu