Isolation and Characterization of an Endophytic Fungus Colletotrichum coccodes Producing Tyrosol From Houttuynia cordata Thunb. Using ITS2 RNA Secondary Structure and Molecular Docking Study

An endophytic fungus isolated from healthy leaf tissues of Houttuynia cordata Thunb., an ethnomedicinal plant of North East India, showed a considerable amount of antimicrobial activity. The ethyl acetate extract of the fungal culture filtrates displayed promising antimicrobial activity against a panel of clinically significant pathogens including Candida albicans, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Bioassay guided purification of the organic extract using column and thin layer chromatography yielded a pure homogenous compound which was identified using spectroscopic methods (essentially by 1H NMR and MS) as tyrosol, a well-known phenylethanoid present in several natural sources. Besides, molecular docking studies against tyrosyl tRNA synthetases (TyrRS) of S. aureus (PDB ID: 1JIL) and E. coli (PDB ID: 1VBM), and CYP45014α-lanosterol demethylase (CYP51) of C. albicans (PDB ID: 5FSA) revealed tyrosol has a strong binding affinity with the enzyme active site residues. The fungus was identified as Colletotrichum sp. and characterized by its genomic ITS rDNA and ITS2 sequences. Phylogenetic analyses showed clustering of our isolate with Colletotrichum coccodes. Species of Colletotrichum are also reported to be plant pathogens. Therefore, to confirm the endophytic lifestyle of the isolate, ITS2 RNA secondary structure study was undertaken. The result indicated our isolate exhibited differences in the folding pattern as well as in motif structures when compared to those of pathogenic C. coccodes. The findings indicated that endophytic fungi harboring H. cordata could be explored as a potent source of antimicrobial agents.

FUNGAL PATHOGENS OF TOMATO IN SOUTH-WESTERN RUSSIA (KRASNODAR TERRITORY)

During a study of fungal diseases of tomato in the South of Russia (Krasnodar Territory) 56 fungal isolates associated with tomato fruits were obtained. Most of them belonged to the species Alternaria alternata. Alternaria solani, Fusarium equiseti, Phomopsis phaseoli, Chaetomium cochliodes, Clonostachys sp., Irpex lacteus, Colletotrichum coccodes were also identified. Laboratory experiments revealed that Clonostachys sp., C. сochliodes, P. phaseoli, I. lacteus, and F. equiseti developed well on the fruit’s slices. Fusarium equiseti was the only species that can penetrate the tomato through epidermis and infect entire fruit. The most effective fungicide against F. equiseti was difenoconazole (EC50 = 0.08 mg/L); pencycuron was also effective (EC50 = 32.5 mg/L). Thiabendazole completely inhibited the growth of F. equiseti at the concentration 100 mg/L (EC50 = 47 mg/L).

Pengaruh Asam Askorbat terhadap Pertumbuhan Colletotrichum coccodes Penyebab Antraknosa pada Tanaman Cabai Merah

Patogen Colletotrichum coccodes merupakan salah satu patogen yang dapat menginfeksi tanaman cabai dan menyebabkan penyakit antraknosa terutama pada bagian buah dan daun. Tanaman dengan kandungan asam askorbat tinggi memiliki ketahanan yang lebih tinggi terhadap serangan patogen. Tanaman cabai yang tahan memiliki kandungan asam askorbat yang lebih tinggi dibandingkan dengan tanaman cabai toleran maupun rentan. Tujuan penelitian ini adalah mengetahui kemampuan tumbuh patogen C. coccodes pada medium yang diberi asam askorbat dan mengetahui pengaruh inokulasi patogen C. coccodes terhadap kandungan asam askorbat pada daun cabai. Penelitian dilakukan di Laboratorium Mikologi dan Fitopatologi, Laboratorium Lingkungan, & Greenhouse Fakultas Biologi Universitas Jenderal Soedirman, Purwokerto. Penelitian ini menggunakan dua uji yaitu uji in vitro dan uji in planta dengan metode eksperimental Rancangan Acak Lengkap (RAL), Uji in vitro menggunakan A) Medium PDA diberi asam askorbat; B) Medium PDB diberi asam askorbat dengan perlakuan penambahan asam askorbat sebanyak 0 mg.L-1 (kontrol); 0,25 mg.L-1; 0,50 mg.L-1; 0,75 mg.L-1; dan 1 mg.L-1, diulang sebanyak lima kali. Variabel bebas yang digunakan yaitu berbagai dosis asam askorbat, variabel terikatnya adalah pertumbuhan patogen C. coccodes. Parameter utama yaitu diameter koloni dan bobot kering miselium. Uji in planta menggunakan tiga varietas cabai (V1: Cabai merah hot chili; V2 Cabai merah keriting; V3: Cabai merah besar), uji A) Uji intensitas penyakit pada tanaman dan B) Uji kandungan asam askorbat. Masing-masing kelompok uji diulang sebanyak lima kali. Variabel bebas yang digunakan adalah varietas cabai merah, variabel terikatnya adalah nilai kerusakan tanaman berdasarkan kategori yang diamati pada waktu pengamatan yang ditentukan. Parameter utama yaitu intensitas penyakit, dan parameter pendukung yaitu periode masa inkubasi, kandungan asam askorbat pada daun cabai, temperatur, kelembaban dan pH tanah. Data uji in vitro dan uji in planta yang diperoleh dianalisis menggunakan analisis ragam (ANOVA) dengan tingkat kepercayaan 95%, dan perlakuan yang memberikan perbedaan nyata dilanjutkan dengan Uji Beda Nyata Jujur (BNJ) pada tingkat kepercayaan 95%. Hasil penelitian pada uji in vitro menujukkan bahwa patogen C. coccodes memiliki kemampuan tumbuh yang baik pada medium PDA dan medium PDB dengan penambahan asam askorbat. Hasil penelitian pada uji in planta, inokulasi patogen C. coccodes pada daun cabai merah dapat meningkatkan kandungan asam askorbat pada tanaman cabai merah Kata kunci : Colletotrichum coccodes, Cabai Merah, Antraknosa, Asam askorbat.

Transcriptome analysis of Phytophthora infestans and Colletotrichum coccodes in tomato to reveal resistance mechanisms

Tomato is considered as an important commercial crop and protective food and it’s yield is extremely affected by different pathogens. Such harmful pathogens severely affecting tomato include Phytophthora infestans (oomycete) and Colletotrichum coccodes (fungi). In this study, gene expression analysis was performed using the microarray datasets available in the public database to investigate differentially expressed genes DEGs under developmental and anatomy conditions and consequently constructing the predicted PPI (Protein-protein interaction) network. Here, a comparison of abundances revealed that 51 genes were differentially expressed due to oomycete and fungi stresses in tomato. Expression analysis revealed that Solyc06g007510.3 was up-regulated whereas Solyc05g008850.3 was down-regulated in all developmental stages and anatomy conditions to manage auxin metabolism under fungi and oomycete infections in tomato. The gene ontology included cellular metabolic and organic substance metabolic processes, cellular biosynthetic, organic substance biosynthetic, and cellular macromolecule metabolic process, chloroplast, intracellular membrane-bounded organelle, heterocyclic compound binding, organic cyclic compound binding, ion binding, binding, and catalytic activity. Analysis of cis-elements illustrated that 12 most general cis-regulatory elements (CATT-box, TATA-box, MYB, MYC, ABRE, G-box, Box 4, AAGAA-motif, MBS, as-1, TGACG-motif, and CGTCA-motif) were identified in promoter region. The highest frequency of cis-elements included CATT-box and TATA-box in response to the pathogens at different growth stages in tomato. Our findings can provide a better insight to the regulatory mechanisms involved in response to fungi and oomycete infection in tomato which can aid to improve the efficiency of developing tomato varieties.

Response of tomato seedlings inoculated with mycorrhizal fungi on the photosynthetic activity, growth, and health status of plants after infection with the fungus Colletotrichum coccodes

The aim of this study was to estimate the effect of mycorrhizal fungi (MF) on the photosynthetic activity, growth, and health status of tomato seedlings (‘Pelikan F₁’) infected with the pathogenic fungus <em>Colletotrichum coccodes</em>. A commercial mycorrhizal inoculum (Mycoflor, Poland) containing spores and dormant mycelium of MF was used in the experiment. It was carried out in a growth chamber where 1-week-old seedlings were inoculated with 3 mL of mycorrhizal inoculum applied into the soil. Three-week old mycorrhized and nonmycorrhized tomato seedlings were infected with the pathogenic fungus. Seedlings planted in sterile horticultural soil without the mycorrhizal inoculum constituted an absolute control. The growth, disease index, and photosynthetic activity of the plants were measured after 4 weeks. There was no significant effect of the mycorrhiza on the photosynthetic parameters analyzed. However, it was noted that the mycorrhized and pathogen-infected seedlings had higher maximum quantum yield of PSII (F_v/F_m), higher effective quantum yield (Y) and more favorable photochemical (qP) and nonphotochemical fluorescence quenching (qN) coefficients than did the pathogen-infected seedlings. The experiment showed that the mycorrhizal commercial inoculum had varied effects on the health status of tomato seedlings depending on the pathogenic fungus isolate. Mycorrhiza did not have a significant effect on the length of roots and stems, whereas the roots inoculated with MF were better developed than those infected with the pathogenic fungus. It can be assumed also that not only the pathogen but also the mycorrhiza is a stress factor towards the seedlings and affects the growth performance parameters mentioned above.