Promising biocontrol agents isolated from medicinal plants rhizosphere against root-rot fungi

Author’s Accepted Manuscript Promising biocontrol agents isolated from medicinal plants rhizosphere against root-rot fungi Moustafa M. Zohair, Ahmed A. El-Beih, Mahmoud W. Sadik, Eman R. Hamed, Mohamed Z. Sedik www.elsevier.com/locate/bab

PII: DOI: Reference:

S1878-8181(18)30188-9 https://doi.org/10.1016/j.bcab.2018.04.015 BCAB751

To appear in: Biocatalysis and Agricultural Biotechnology Received date: 25 February 2018 Revised date: 19 April 2018 Accepted date: 25 April 2018 Cite this article as: Moustafa M. Zohair, Ahmed A. El-Beih, Mahmoud W. Sadik, Eman R. Hamed and Mohamed Z. Sedik, Promising biocontrol agents isolated from medicinal plants rhizosphere against root-rot fungi, Biocatalysis and Agricultural Biotechnology, https://doi.org/10.1016/j.bcab.2018.04.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Promising biocontrol agents isolated from medicinal plants rhizosphere against root-rot fungi Moustafa M. Zohaira*, Ahmed A. El-Beiha, Mahmoud W. Sadikb, Eman R. Hameda, Mohamed Z. Sedikb a

Chemistry of Natural and Microbial Products Department, Pharmaceutical Industries Research Division, National Research Centre, Giza, 12311, Egypt. b Microbiology Department, Faculty of Agriculture, Cairo University, Egypt. *

Corresponding author: [email protected]

Abstract Among of the collection of 104 fungal isolates from the rhizosphere of the medicinal plants basil (Ocimum basilicum), peppermint (Mentha piperita) and (Aloe vera), 59 of them were assessed for in vitro antagonistic activity against phytopathogenic fungi; Fusarium solani, Rhizocotina solani, Sclerotium rolfsii and Verticillium dahliae. The most active antagonistic isolates were identified using molecular tools based on 18S rDNA. The sequence data of Aspergillus pseudocaelatus and Trichoderma gamsii have been submitted to GenBank given the accession no. MG772677 & KX685665, respectively. The antagonistic mechanisms were evaluated using confrontation method and scanning electron microscopy. The volatile organic compounds (VOCs) were analyzed using GC/MS. Biological investigation on the ethyl acetate extract of A. pseudocaelatus MG772677 and T. gamsii KX685665 isolates was evaluated. The greenhouse application with the selected isolates on basil seedlings was performed. In vitro antagonistic results indicated that the highest percentage of inhibition was observed with A. pseudocaelatus, 77.90 % and T. gamsii, 77.98%. The average emergence rate in the treatments with T. gamsii and A. pseudocaelatus reached up to 100%, much more than that in the control (average 40%). The results indicated that A. pseudocaelatus MG772677 and T. gamsii KX685665, displayed antagonistic activities against the pathogenic fungi and presented appreciable biocontrol efficacy. Also the two isolates could enhance the plant growth and improve the seedlings’ emergence.

Keywords: Aspergillus pseudocaelatus; Trichoderma gamsii; mycoparasitism; VOC; biocontrol; phytopathogenic fungi 1. Introduction Microorganisms have been proved to be sources of natural compounds for many industries including pharmacy and agriculture. Microorganisms as biocontrol agents have high potential application to control phytopathogens with no adverse effect on the environment or other non-target organisms (Khamna et al., 2009). Soil-borne pathogens cause economical losses in agricultural products, fungi being the most aggressive. The distribution of several phytopathogenic fungi, as 1

Fusarium, Pythium, Phytophthora, Botrytis and Rhizoctonia, has spread during the last few years due to changes applied in farming (Benítez et al., 2004). Biocontrol of plant pathogens in addition plant growth enhancement with microbes have been screened over the last years and are becoming an applicable alternative to fertilizers and chemical pesticides in sustainable agriculture (Weller, 2007). Indirectly, the plant growth could be enhanced by biocontrol of plant pathogens thorough antibiotics or volatile organic compounds (VOCs), toxins, bio-surfactants, or cell wall-lytic enzymes, but antagonistic activities of microbes could also happen through degradation of pathogenicity factors such as toxins, or the competition for minerals, nutrients, or colonization positions (Berg, 2009). Fungi have supplied a plenty of bioactive compounds currently used as pharmaceuticals and biopesticides. The soil is basically the main fungal genetic resource for bioprospection programs (Adrio and Demain, 2003). However, the diversity and biotechnological potential of the soil mycobiota in several tropical regions need more studies. Butler (2004) reported that over 40 % of medicines used today were fungal metabolites. A huge number of the compounds isolated from soil fungi present an effective biological tools that can be applied as antifungal, antibacterial and antitumor in drug discovery programs (Sadiran, 2011). The well-known fungal genera Aspergillus, Fusarium, Penicillium, Piriformospora, Phoma, and Trichoderma were the most frequently reported plant growth promoting fungi (PGPF). PGPF were generally believed to be beneficial for all plant species they associate with, because of their conserved beneficial abilities. PGPF directly and indirectly influence the growth and productivity of a wide range of host plants (Hossain et al., 2017). The biotechnological potential of Aspergillus relies not only on economic relevance of microbial fermentations, but also on the genomic diversity leading to the production of several bioactive metabolites, classifying the genus as an attractive and valuable source for drug discovery (Lee et al., 2013). Aspergillus is one of the most intensely studied fungal genera with respect to production of secondary metabolites. Members of this genus of fungi are ubiquitous in nature and are capable of living as saprophytes in soils (Geiser et al., 2007). In recent years, the filamentous fungi Trichoderma spp. (teleomorph Hypocrea) have attracted the research attention due to their activity as a biological control agents against several phytopathogens (Harman et al.,2004; Dubey et al.,2007; Hermosa et al.,2012; Sriram et al., 2013). Trichoderma species have an effect on pathogens thorough mycoparasitism, excreting cell wall-degrading enzymes, antimicrobial compounds directly, or indirectly affect the phytopathogens by competing for nutrient supplies and sites, modifying the environmental conditions, enhancing plant growth, an indirect systemic defence response to phytopathogen (Benítezet al., 2004; Vos et al., 2015), as well as increasing the availability of nutrient. Trichoderma volatile organic compounds are reportedly promoting plant growth (Hung et al., 2013; Lee et al., 2015). Also, 2

Trichoderma are able to interact with the pathogens inactivating the pathogenicity related proteins. More than 200 bioactive compounds have been discovered from Trichoderma species, producing compounds that present promising biocontrol effects (Reino et al., 2008; Vinale et al., 2013). Viridepyronone extracted from the cultural filtrate of Trichoderma viride had an antagonistic effects against S. rolfsii in vitro (>90% inhibition) (Evidente et al., 2003). The pyrone 6-pentyl-2H-pyran-2-one, the most common metabolite produced by Trichoderma was tested in confrontation activities against Fusarium oxysporum and R. solani ( Reino et al., 2008). Medicinal plants harbor a unique microbiome because of their distinctive and structurally divergent metabolites that are usually responsible for the associated microorganisms specificity (Qi et al., 2012). Medicinal plants are a largely undiscoverable and great bio resource for bioactive compounds and microorganisms of potential agricultural, medical and pharmaceutical applications (Köberl et al., 2013). A specific strategy of biocontrol for medicinal plants, which are increasingly affected by several soil-borne plant pathogens, has not been available until now. While specific biological control for medicinal plants are needed, their associated microorganisms with outstanding metabolic effects also represent a promising source for new biocontrol agents (BCAs) (Köberl et al., 2013). The aim of this research was to select the most active fungal isolates from medicinal plants rhizosphere as potent biocontrol agents against root-rot fungi.

2. Materials and Methods 2.1. Isolation, purification and identification of rhizospheric fungi Fresh, healthy parts of roots of three medicinal plants basil (Ocimum basilicum), peppermint (Mentha piperita) and

(Aloe vera), cultivated on organically managed

Egyptian desert farm, Sekem, Heliopolis University, Cairo, Egypt have been cut into pieces and placed in Czapek yeast extract agar (CYA) containing chloramphenicol and streptomycin to control bacterial growth. After one week incubation at 28°C, when mycelium tips of fungi were observable, each tip was transferred into a PDA slant. For purification of the fungal strains, this step was repeated until the colony was deemed uniform. The pure isolates were numbered and stored on PDA slants at 4° C. (Guraud, 1998).

3

Fungal characterizations were done according to their macro and microscopic characteristics. The cultural and morphological characteristics of the isolates were compared with species descriptions reported in the standard taxonomic guidelines to identify the fungal genera and species (Hanlin, 2000).

2.2.In vitro antagonism test of isolated fungi Fifty nine isolates were evaluated in vitro as antagonists against local isolates of F. solani, R. solani, S. rolfsii and V.dahliae obtained from the culture collection of the Chemistry of Natural and Microbial Products Department, National Research Centre, (NRC) Cairo, Egypt. Dual cultures technique was applied by using one week-old cultures of soil borne pathogenic fungi and fungal isolates grown on PDA medium. Five-mmdiameter disc of each isolate was suspended in 5 ml sterilized distilled to prepare the inoculum. PDA medium was inoculated with 1 ml of prepared inoculum and allowed to solidify. After solidification, 6-mm-diameter disc of the pathogen was placed in the center of the dish. Fungi were incubated for 7 days at 28°C. Controls were performed using pathogens against itself. There were three replicates for each treatment. At the end of the incubation period, radial growth was measured and inhibition percentage was calculated as follow: Inhibition Percentage = (R1-R2)/R1×100 Where R1; diameter growth of the pathogen in control and R2; diameter growth of the pathogen in dual culture with antagonist (Satish et al., 2007; Bouzaine et al., 2011). 2.3. Genotypic identification of the most active fungal isolates 2.3.1. DNA extraction, PCR amplification, sequencing and molecular phylogenetics Based on the antagonism results; two isolates representing the highest antagonistic activities were selected and identified using molecular tools based on 18S rDNA. The selected fungal isolates were inoculated individually into 20 ml potato dextrose broth (PDB) medium.

Mycelium was harvested and DNA was extracted

according to Abd-Elsalam et al., 2007. The fungal inter transcribed spacer (ITS) fragments were amplified by PCR using the universal primers ITS1 and ITS4. The 4

sequences of the ITS1 and ITS4 primers were 5′-TCCGTAGGTGAACCTGCGG-3′ and 5′-TCCTCCGCTTATTGATATGC-3′

(White

et

al.,

1990),

respectively.

PCR

amplification was done according to the following temperature profile: an initial step of 2 min at 94 °C, 40 cycles of 60 s at 94 °C, 90 s at 52 °C and 2 min at 72 °C, and a final step of 7 min at 72 °C. The PCR products were analyzed by agarose gel electrophoresis and purified using kit (AccuPrep PCR DNA Purification Kit, K-3034-1, Bioneer Corporation). The purified PCR products were sequenced with Macrogen Inc. (Seoul, South Korea), all inter transcribed spacer sequencing work was also done by Macrogen and was applied on both strands of the submitted DNA fragments. The sequences were assembled, edited and aligned by the DNA STAR SeqMan (DNASTAR Inc.) and the CLC sequence viewer.

2.4. Liquid culture and metabolite production Two 6-mm diameter plugs of A. pseudocaelatus MG772677 and T. gamsii KX685665 isolates, obtained from growing margins of PDA cultures, were inoculated into 3 L conical flasks containing 1L of sterile potato dextrose broth (PDB), individually. The flasks were incubated statically for 15 days at 28°C. The cultures were filtered through filter paper (Whatman No. 4; Brentford, UK). 900 ml of the culture filtrate was stored at 2 °C for use in the greenhouse experiment (Vinale et al., 2006). The other 100 ml of the culture filtrate was partitioned with ethyl acetate for three successive times. The ethyl acetate layer was concentrated under vacuum to obtain the ethyl acetate extract for each fungus to be used in GC/MS and antimicrobial experiments.

2.5. Determination of volatile organic compounds (VOCs) by GC/MS The

GC-MS

system

(Agilent

Technologies)

was

equipped

with

gas

chromatograph (7890B) and mass spectrometer detector (5977A) at Central Laboratories Network, National Research Centre (NRC), Cairo, Egypt. The GC used column was HP5MS (30 m x 0.25 mm internal diameter and 0.25 μm film thickness). The helium as carrier gas at a flow rate of 1 ml/min was used at a split ratio of 1:5, injection volume was 1 µl of ethyl acetate fungal extract and the temperature program: 70 °C for 2 min; rising at 4 °C/min to 180 °C and held for 1 min; then rising at 6 °C/min to 260 °C and held for 5 5

min. The injector and detector were held at 280 °C and 270 °C, respectively. Mass spectra were determined by electron ionization voltage (EI) at 70 eV; using a spectral range of m/z 35-550 and solvent delay 2 min. The metabolites were identified based on Wiley and NIST Mass Spectral Library data (Adams, 1995). 2.6. Evaluation of antagonistic potential 2.6.1.Dual culture interaction examination by scanning electron microscope (SEM) The method of direct confrontation consisted to put one agar disc (6 mm diameter) of the isolate and another one of the same diameter representing the pathogen on the same Petri dish containing 15ml of PDA medium. They were both placed along a diametrical axis 3cm away. Incubation is done at 28°C for 3-5 days in the dark. The mycoparasitism of T. gamsii was studied by harvesting the hyphae from the contact zone between T. gamsii and the phytopathogens. The hyphal sample was transferred to glass coverslips, fixed with 2.5% glutaraldehyde, dehydrated in a series of ascending ethanol concentrations (50-100%) (v/v), dried in desiccator, and coated with gold in a sputtercoater (SCD 005; BAL-TEC, Switzerland); then, The examination was done by scanning electron microscope (Quanta 250 FEG, FEI Company, USA) (Barak et al., 1986). 2.7. Hydrolytic enzyme detection Preliminary screening of enzymatic activities of A. pseudocaelatus and T. gamsii strains were tested for hydrolysis. The hydrolytic activities of chitinase and cellulase were tested according to Kasana et al., 2008; Sharma and Salwan, 2015. The enzyme activity percentage was determined using the following formula as: Capacity of hydrolysis (%) = [A/B]*100 Where A; diameter of clear zone (cm) occurred by grown isolate on media contained carboxymethyl cellulose (CMC) or chitin as carbon sources, B; Diameter of isolate colony (cm) grown on PDA media (Saravanakumar et al., 2017). 2.8. Evaluation of antimicrobial activities of the crude fungal extract

6

The ethyl acetate fungal extract (1mg/5 μl) was assessed for its antimicrobial activity using agar diffusion method (El-Sawy et al., 2015) against fungi (F. solani, R. solani, S. rolfsii and V. dahliae), yeast (Candida albicans ATCC10321, Candida tropicalis ATCC750), Gram positive bacteria (Bacillus subtilis ATCC 6633 and Staphylococcus aureus ATCC 25923) and Gram negative bacteria (Escherichia coli ATCC 25922 and Salmonella enteric ATCC 25566). Bacteria and yeast strains were obtained from the American Type Culture Collection while fungi were obtained from the culture collection of the Department of Chemistry of Natural and Microbial Products, National Research Centre, Egypt. Thiophenicol (Thiamphenicol, Sanofi aventis, France) and Treflucan (Fluconazole, Egyptian Pharmaceutical Industries Company EIPICO) were used as antibacterial and antifungal positive controls, respectively, in a concentration of 100 μg/disc and DMSO was used as a negative control. The discs were applied on inoculated nutrient agar and PDA media plates and incubated for 24 h at 30 °C for bacteria and 72 h at 28 °C for fungal strains, respectively. The zone of inhibition was recorded and compared with the positive control treatments. 2.9. Greenhouse Experiment The biocontrol efficacy of evaluated isolates was estimated with detached healthy basil seeds. Infection technique was applied using one week-old soil borne pathogenic fungi cultures on PDA. Five ml sterilized distilled water suspension was inoculated with a 5-mm diameter disc of soil borne pathogenic fungi to prepare the inocula. To make artificial infection, the prepared inocula were added into pots (each containing 400 g sterilized soil previously autoclaved at 121°C for 15 min). The treatments were designed as follows Table 1 Greenhouse experiment design Seeds soaked in Treatment

NC*** P PC**** T1 T2 T3 T4

Sterile water

Fungicide**

+ + -

+ -

Culture filtrate T. A. gamsii pseudocaelatus + + + +

Biocontrol agent Pathogenic fungi + + + + + +

Agricultural waste*

T. gamsii

A. pseudocaelatus

Rice straw

Olive

+ + +

+ -

+ -

+

7

T5 T6

+ + + + + + + *Agricultural waste (25 g) previously autoclaved and inoculated with the biocontrol fungus for two weeks at 28 ºC. ** Fungicide; RIZOLEX- T50% WP, Sumitomo Chemical LTD, Japan in concentration 1g/l. *** Negative control. **** Positive control.

+

Each treatment was done in triplicate. At the end of experiment, the vegetative growth parameters were estimated. Disease index (DI) was estimated on scale 0-4 according to Harveson et al., 2014, the statistical design was completely randomized. 2.10. Statistical Analysis The data were subjected to ANOVA and were analyzed by MSTATC program. The differences of means of three replicates were compared using LSD test at 5% level of significance. 3. Results and Discussion 3.1. Microorganisms isolation Around 104 fungal isolates belonging to 15 genera: Absidia, Alternaria, Aspergillus, Botrytis, Emericella, Eurotium, Fusarium, Humicola, Mucor, Rhizoctonia, Rhizopus, Sclerotium, Scopulariopsis, Trichoderma and Verticillium were isolated from the rhizosphere of three medicinal plants (basil Ocimum basilicum, peppermint Mentha piperita and Aloe vera). 3.2. Identification of the most active isolates based on molecular phylogenetic analysis The fungi were identified as A. pseudocaelatus and T. gamsii (Fig. 1) based on morphological, microscopic features and 18S rDNA sequence (ITS phylogenetic analysis). The sequence data have been deposited at GenBank (accession no. MG772677 & KX685665, respectively).

8

Fig.1. Phylogenetic tree showing the relationship of A. pseudocaelatus and T. gamsii with previously reported isolates of Aspergillus spp. and Trichoderma spp

3.3. Dual culture and mycoparasitism The percentage of inhibition observed with A. pseudocaelatus was 71.43, 80.17, 82.64 and 77.36 % against the phytopathogens F. solani, R. solani, S. rolfsii and V. dahliae, respectively. While T. gamsii exhibited the inhibition percentages 74.29, 77.16, 81.06 and 79.43% against the phytopathogens F.solani, R. solani, S. rolfsii and V. dahliae, respectively. (Fig. 2). Hamed et al. (2012) observed that during hyphal interactions, direct growth of the T. harzianum against the hyphae of R. solani had occurred accompanied by an enhanced branching by the mycoparasite and an abnormal growth of the target hyphae. Cundom et al. (2000) observed the ability of T. harzianum to invade the colony of the pathogen thorough in vitro competition between the antagonist

Inhibition %

against Sclerotina sclerotium. 90 80 70 60 50 40 30 20 10 0

71.43

74.29

80.17

77.16

82.64

81.06

77.36

79.43

A.pseudocaelatus MG772677 T. gamsii KX685665

F. solani

R. solani

S. rolfsii Phytopathogens

V. dahliae

9

Fig.2. Antagonistic activities of A. pseudocaelatus and T. gamsii against phytopathogenic fungi; F. solani, R. solani, S. rolfsii and V. dahliae.

As shown in (Fig. 3) scanning electron microscopy examination of T. gamsii hyphae in the contact zone, displayed that hyphal interactions exist, which could demonstrate a parasitic effect of T. gamsii on F. solani. It envisioned the growth surrounding the T. gamsii hyphae on F. solani (Fig. 3 FT), wrapping them either tightly or loosely. Also, it was observed that the hyphae of T. gamsii grew in parallel to hyphae of F. solani normally. The shrinking in the mycelia of R. solani were related to cytoplasmic disorganization, retraction and rupture of the plasma membrane leading to alteration and deformation of the cell wall in the site of penetration of the antagonist. All these effects may cause cellular lyses as shown in (Fig. 3 RT). These results is in agreement with (Cuervo-parra et al., 2011) who observed that during the confrontation period in dual culture, the hyphae of T. harzianum grew on the hyphae of Moniliophthora roreri, causing morphological deformations and disorganization in the structure of their cell wall so that its appearance becomes rough, probably due to the secretion of antifungal substances (enzymes and antibiotics) by T. harzianum. At certain contact points T. gamsii was connected to the pathogenic fungus S. rolfsii with small branches as apresorious (Fig. 3 ST). These observations could clear the particular parasitic activity of the antagonist that finally led to inhibition of the pathogen growth. T. gamsii hyphae grew in parallel to the V. dahliae hyphae and in some sites; these were interacted with the hyphae of the pathogen (Fig. 3 VT). These morphological changes in the structure of the phytopathogenic fungi caused by T. gamsii were similar to those observed by Cuervoparra et al., 2011, who cleared that the disintegration of mycelial walls resulted in the total destruction of M. roreri during the parasitism by T. harzianum. Harman et al. (2004) have reported the ability of Trichoderma spp. parasitism on pathogens by attaching and wrapping of its hyphae around the pathogens. Also, Pates et al. (1999) reported that the Trichoderma viride has an important ability to secrete enzymes to attack or remove mycotoxins produced by the pathogens.

10

Fig.3. Scanning electron micrographs (Quanta 250FEG; FEI Company, USA) examination of hyphal interaction of T. gamsii against F. solani, R. solani, S. rolfsii and V. dahliae (FT, RT, ST and VT), respectively. 3.4. Volatile organic compounds (VOCs) determination by GC-MS The predominant volatile compounds predicted by GC-MS analysis, (Table 2) were 2,3,4-Trimethoxyphenylethylamine, 3-methoxy-2-(1-methylethyl)-5(2methylpropyl) pyrazine, (Z)-9- Octadecenamide, Pyrrolo [1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-, Thieno [2,3-c] pyridine-3-carboxamide,4,5,6,7tetrahydro-2-amino-6-methyl- and Hexadecanamide. Chen et al.(2015) reported that the T. gamsii produces a plenty of VOCs which may inhibit the growth of pathogenic fungi. Hussein et al.(2016) reported that (Z)-9-Octadecenamide compound has an antiinflammatory activity and antibacterial activity. Also, fatty acids such as oleic acid has a potential antibacterial and antifungal activity (Agoramoorthy et al., 2007).The VOC profiles of Trichoderma species include alcohols, ketones, alkanes, furanes, pyrones and terpenes (Stoppacher et al., 2010) which have varying degrees of antagonistic activity against pathogenic fungi.

11

A. Pseudocaelatus MG772677

T. gamsii KX685665

Fig.4. The volatile organic compounds (VOC) profiles produced by T.gamsii and A. pseudocaelatus. Peaks of compounds were recorded and identified in (Table 2).

Table 2 Volatile organic compounds (VOCs) profile by GC/MS. A. pseudocaelatus Volatile compounds Peak no.

RT

Area Sum %

T. gamsii Peak RT no.

Area Sum%

1-Propyl-3,6-diazahomoadamantan-9-ol

1

22.962

0.59

Nil

Nil

Nil

Thieno[2,3-c]pyridine-3-carboxamide, 4,5,6,7-tetrahydro-2-amino-6methyl3,4-Dihydrocoumarin, 6-fluoro-4,4-dimethyl3-methoxy-2-(1-methylethyl)-5-(2-methylpropyl) pyrazine 2,3,4-Trimethoxyphenylethylamine 3-methoxy-2-(1-methylethyl)-5-(2-methylpropyl) pyrazine Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)(1,5,5,8-Tetramethyl-bicyclo[4.2.1]non-9-yl)-acetic acid (1R,4aR,4bR,10aR)-7-Isopropyl-1,4a-dimethyl-1,2,3,4,4a,4b,5,6,10,10adecahydrophenanthrene-1-carbaldehyde Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)Hexadecanamide (Z)-9-Octadecenamide Ergotamine

2

23.262

1.45

Nil

Nil

Nil

3 4 5 6 8 9 10

24.033 25.109 25.592 26.562 29.456 29.739 31.480

0.69 3.59 19.79 3.19 0.93 1.37 3.19

Nil Nil Nil Nil 3 Nil Nil

Nil Nil Nil Nil 31.985 Nil Nil

Nil Nil Nil Nil 2.35 Nil Nil

11 15 18 19 22

32.015 37.415 40.403 40.544 43.055

1.54 1.27 2.1 0.86 49.44

3 10 13 Nil 17

31.985 37.42 40.42 Nil 43.055

2.35 1.99 3.58 Nil 63.35

23 Nil Nil Nil Nil Nil Nil

46.114 Nil Nil Nil Nil Nil Nil

0.67 Nil Nil Nil Nil Nil Nil

Nil 18 5 6 8 9 11

Nil 46.114 32.703 33.268 34.074 36.726 38.097

Nil 1.09 1.04 1.19 0.67 0.82 0.67

Nil

Nil

Nil

14

40.573

1.51

Nil

Nil

Nil

16

41.603

1.63

*

Bis(2-ethylhexyl) phthalate trans-13-Docosenamide 13-Docosenamide, (Z)Tertbutyloxyformamide, N-methyl-N-[4-(1-pyrrolidinyl)-2-butynyl] n-Hexadecanoic acid Dihydroxanthin Oleic Acid 1H-Pyrano[3,4-c]pyridine-5-carbonitrile, 3,4,6,7-tetrahydro-3,3-dimethyl-8(1-pi Ergotaman-3',6',18-trione, 9,10-dihydro-12'-hydroxy-2'-methyl-5'(phenylmethyl)-, (5'.alpha.,10.alpha.) 1-Acetyl-2-amino-3-cyano-7-isopropyl-4-methylazulene

*Phthalate esters are likely contaminants from plastic ware in the laboratory during extraction and isolation. 3.5. Hydrolytic enzyme detection

12

A. pseudocaelatus MG772677 and T. gamsii KX685665 isolates showed a high hydrolytic capacity for carboxymethyl cellulose (CMC) and moderate capacity of hydrolysis of chitin as shown in (Fig. 5). The production of the extracellular cell wall lytic enzymes as cellulase, chitinase, β-(1–3) glucanase and protease of fungi could have an effective role in the inhibition of the pathogenic fungi (Gajera et al., 2012; Vinale et al., 2008) and enhanced resistance of plants. Inoculated plates with A. pseudocaelatus

Inoculated plates with T. gamsii

CMC or Chitin Uninoculated Agar plates

34.29 ±1.42

36.25±6.25

-

62.14 ±2.14

70.00±5

-

Chitin

Hydrolysis capacity %

Carboxymethyl cellulose (CMC)

Hydrolysis capacity%

The values represent the mean of three replicates± S.D.

Fig. 5. Hydrolytic enzymes activities of the A. pseudocaelatusor T. gamsii inoculated on carboxymethyl cellulose (CMC) or chitin agar plates compared with uninoculated plates. 3.6. Evaluation of antimicrobial activities of the crude fungal extract The antimicrobial activity of ethyl acetate extract of A. pseudocaelatus was stronger than of T. gamsii as shown in (Table 3). It showed a potent inhibitory effect against fungal test strains (C. albicans, C. tropicalis, F. solani, R. solani, S. rolfsii and V. dahliae with inhibition zones 13.5, 15.5, 14, 15, 7 and 8 mm diameter, respectively). While for the bacteria test strains, the extract of A. pseudocaelatus has moderate activity against B. subtilis, S. aureus, E. coli and S. enteric with inhibition zone diameter of 20, 19.5, 18.5 and 14.5 mm, respectively. T. gamsii extract showed a moderate effect against all pathogenic test microorganisms in comparison to that of A. pseudocaelatus. Saleh et al. (2011) reported the antibacterial activity of some Trichoderma spp. crude extracts against some human pathogenic bacteria with inhibition zones ranging 13

from 10 to 28 mm. Vinale et al. (2006) reported that the secondary metabolites produced by Trichoderma harzianum strains T22 and T39 have effects against the pathogens R. solani and Pythium ultimum. Table 3 Antimicrobial activity of ethyl acetate fungal extract of A. pseudocaelatus and T. gamsii. Inhibition zone diameter (mm) Pathogenic G+bacteria G- bacteria Yeast Fungi B. S. E. S. C. C. F. R. S. V. Extract subtilis aureus coli enterica albicans tropicalis solani solani rolfsii dahliae A. pseudocaelatus 20±0.0 19.5±0.5 18.5±0.5 14.5±1.5 13.5±0.5 15.5±0.5 14±0.6 15±0.0 7±0.0 8±.01 T. gamsii 12±0.0 13.5±0.5 9±0.0 7±0.0 8±0.0 7±1 8±0.0 7±0.0 7±0.0 7±0.0 Positive control 22.5±0.5 18.5±0.5 11.5±0.5 15±0.0 7.5±0.5 7±0.0 7±0.0 7±0.0 7±0.0 8.5±0.5 Notes: The inhibition zone diameter (Values are mean ± S.D.) expressed in (mm). Thiophenicoland Treflucan were used as positive controls at a concentration of 100 μg/disc, DMSO was used as negative control.

3.7. Plant growth promoting and biocontrol properties The most commonly reported activities of plant growth promoting fungi (PGPF) including the fungal genera Aspergillus and Trichoderma are significant improvement in germination, seedling vigor, biomass production, root hair development, photosynthetic efficiency, flowering, and yield. Some strains have the abilities to improve plant biochemical composition (Hossain et al., 2017). The greenhouse application showed appreciable biocontrol efficacy against root-rot pathogenic fungi. The average shoot length was significantly 23.87 and 8.57 cm with T5 and PC treatments, respectively. The treatment T6 showed the highest shoot fresh and dry biomass (8.08 and 1.095 g, respectively while, the treatment PC presented the lowest values (0.87 and 0.08 g), respectively. The root fresh and dry biomass were 2.039 and 0.87 g of T3 and T4 treatments, respectively. The germination percentage of T3 treatment was the highest significantly. Murali et al. (2012) reported that the plant growth promoting fungus (Trichoderma sp. UOM 37) offered maximum germination of 91% and seedling vigor of pearl millet under greenhouse condition. The disease index varied significantly between the treatments of T3 and PC (0 and 2.8), respectively as shown in (Table 4). These results indicate that, the treatment with T. gamsii may promote the root growth and enhance the induced systemic resistance of the host plant to root rot fungi. Similar results are also reported in the work of

Saravanakumar et al., 2017, who revealed that

exogenous inoculation of Trichoderma harzianum strain CCTCC-RW0024 could significantly enhance the plant growth, plant biomass and reduce the pathogen invasion. 14

Also, Marra et al. (2006) reported that some Trichoderma species utilized as biocontrol agents are able to interact simultaneously with plants and pathogenic fungi, thus can be used as model microorganisms to analyze complex and multiple-player plant-microbe interactions. These species could be applied in agriculture because of their capability to parasitize on several pathogenic fungi such as Botritis spp., Fusarium spp., Pythium spp., Rhizoctonia spp. and Sclerotinia spp. Trichoderma species could also be widely applied for their biocontrol activities (Jelen et al., 2013). Saravanakumar et al. (2017) reported that BCAs of Trichoderma spp. have ultimate roles in promoting the plant beneficial microbial community and reducing the pathogen invasion through the specific interactions with host pathogen. During the mycoparasitism, phytopathogen cell walls are degraded by cell wall lytic enzymes produced by Trichoderma (Reithner et al., 2011).

Trichoderma gamsii T6085 contains a large number of specific enzymes (CAZy), such as GH2,GH30, GH13, and GH5, ceratoulmin hydrophobin, proteinase inhibitor, peptidases, such as S53 and G1 and glucose/ribitol dehydrogenase enzymes (Baroncelli et al., 2016). T. gamsii isolate also showed antagonistic activity to phytopathogens (Aydin and Turhan, 2013; Gilardi et al., 2015). The recognition process was thought as the precondition necessary to inhibit the pathogens during the mycoparasitism that happened between Trichoderma and pathogens (Benhamou and Chet, 1993; López-Mondéjar et al., 2010). Table 4 Effect of different treatments on biocontrol properties and plant growth promoting. Treatment

Shoot Leaves Stem Shoot fresh Shoot dry Root fresh Root dry Germination Disease length no. diameter weight (g.) weight weight (g.) weight % Index (DI) (cm.) (mm.) (g.) (g.) NC 15.30 BC 9.0 C 0.23 BC 2.63 BCD 0.220 C 0.171 D 0.066 B 80 A-D 1.00 B PC 08.57 D 8.0 C 0.19 C 0.87 D 0.080 C 0.207 D 0.113 B 69.97 A-D 2.80 A P 10.56 D 9.7 C 0.24 BC 1.53 D 0.147 C 0.294 CD 0.144 B 71.65 A-D 2.32 A T1 11.57 CD 8.8 C 0.23 BC 2.53 CD 0.224 C 0.585 BCD 0.301 AB 58.3 CD 1.15 A T2 10.72 D 11.80 BC 0.21 C 2.74 BCD 0.436 BC 0.814 BCD 0.326 AB 50 D 2.50 A T3 16.87 B 16.05 B 0.31 A 4.70 BC 1.254 A 2.039 A 0.516 AB 100 A 0D T4 18.37 B 15.95 B 0.32 A 4.86 B 0.400 BC 1.509 ABC 0.871 A 89.97 ABC 0.47 BCD T5 23.87 A 23.77 A 0.33 A 7.66 A 0.908 AB 1.665 AB 0.766 A 93.32 AB 0.22 CD T6 23.73 A 25.22 A 0.28 AB 8.08 A 1.095 A 1.513 ABC 0.767 A 61.65 BCD 0.90 BC LSD %5 4.211 4.549 0.064 2.237 0.6371 1.225 0.607 34.29 0.734 Values are mean and means in a given column with the same letters are not different significantly (˛ > 0.05). NC; negative control,

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PC; positive control, cultivated pots with soaked seeds in fungicide, inoculated with pathogenic fungi and treated with fungicide, P; cultivated pots with soaked seeds in sterilized water, inoculated with pathogenic fungi without any treatment.T1; Cultivated pots with seeds, inoculated with pathogenic fungi and treated with the culture filtered of T. gamsii, T2; Cultivated pots with the seeds inoculated with pathogenic fungi and treated with the culture filtered of A. pseudocaelatus, T3 and T4; Cultivated pots containing sterilized soil and 25 g. of agricultural waste (rice straw or olive waste) inoculated and incubated for 2 weeks with T. gamsii and T5 and T6; Cultivated pots containing sterilized soil and 25 g. of agricultural waste (rice straw or olive waste) inoculated and incubated for 2 weeks with A. pseudocaelatus.

4. Conclusion In vitro antagonisms results indicated that the highest percentage of inhibition observed with A. pseudocaelatus and T. gamsii which was 77.90 and 77.98 %, respectively. Biocontrol experiment showed that the seedlings inoculated with T. gamsii, A. pseudocaelatus and the other four pathogenic fungi remained healthy during the observation period. The inoculation of T. gamsii could significantly increase the plant growth, plant biomass and reduce the pathogen attack. Also, T.gamsii produces a plenty of VOCs which may inhibit the growth of pathogenic fungi. The associated microbiomes of medicinal plant could provide a promising source for novel BCAs. Acknowledgments The authors acknowledge the National Research Centre (NRC), Egypt for funding the research project entitled (Production of some metabolic byproduct compounds which have biological activity from rhizospheric fungi) (No.11010103). Conflict of interest The authors declare that they have no conflict of interest. Appendix A. Supporting information Supplementary data associated with this article available as an attached file References Abd-Elsalam, K., Asran-Amal, A., El-Samawaty A., 2007. Isolation of high quality DNA from cotton and its fungal pathogens. Journal of Plant Protection and Diseases 114,113-116. Adams, R. P., 1995. Identification of essential oils components by Gas Chromatography/Mass Spectroscopy, Allured publishing Cooperation, Carol Stream, Illinois, USA. Adrio, J. L., Demain, A. L., 2003. Fungal biotechnology. International Microbiology, 6, 3, 191-199.

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