In vitro Antagonistic Activity of Endophytic Fungi Isolated from Shirazi Thyme (Zataria multiflora Boiss.) against Monosporascus cannonballus

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VOLUME 69 , ISSUE 3 (Sep 2020) > List of articles

In vitro Antagonistic Activity of Endophytic Fungi Isolated from Shirazi Thyme (Zataria multiflora Boiss.) against Monosporascus cannonballus

RAHIL SAID AL-BADI / THAMODINI GAYA KARUNASINGHE / ABDULLAH MOHAMMED AL-SADI / ISSA HASHIL AL-MAHMOOLI / RETHINASAMY VELAZHAHAN *

Keywords : Zataria multiflora, antifungal, endophytic fungi, Monosporascus cannonballus

Citation Information : Polish Journal of Microbiology. Volume 69, Issue 3, Pages 379-383, DOI: https://doi.org/10.33073/pjm-2020-029

License : (CC-BY-4.0)

Received Date : 23-March-2020 / Accepted: 18-June-2020 / Published Online: 05-August-2020

ARTICLE

ABSTRACT

Endophytic fungi viz., Nigrospora sphaerica (E1 and E6), Subramaniula cristata (E7), and Polycephalomyces sinensis (E8 and E10) were isolated from the medicinal plant, Shirazi thyme (Zataria multiflora). In in vitro tests, these endophytes inhibited the mycelial growth of Monosporascus cannonballus, a plant pathogenic fungus. Morphological abnormalities in the hyphae of M. cannonballus at the edge of the inhibition zone in dual cultures with N. sphaerica were observed. The culture filtrates of these endophytes caused leakage of electrolytes from the mycelium of M. cannonballus. To our knowledge, this is the first report on the isolation and characterization of fungal endophytes from Z. multiflora as well as their antifungal effect on M. cannonballus.

Graphical ABSTRACT

The term “Endophytes” denotes microorganisms that colonize plants’ internal tissues for part of or throughout their life cycle without producing any apparent adverse effect. The endophytic microorganisms include fungi, bacteria, and actinobacteria (Bacon and White 2000). Among them, fungi are the most common endophytic microorganisms (Staniek et al. 2008). Endophytic fungi are ecologically distinct polyphyletic groups of microorganisms, mostly belonging to the Ascomycota phylum (Jia et al. 2016). Several fungal endophytes have been shown to act as biological control agents for managing soil-borne plant pathogens (Toghueo et al. 2016).

Zataria multiflora Boiss. (Synonyms: Zataria bracteata Boiss.; Zataria multiflora var. elatior Boiss), belonging to the Lamiaceae family is a traditional medicinal plant commonly used as a flavor ingredient in different types of foods (Sajed et al. 2013). Several medicinal properties of Z. multiflora, including antiseptic, anesthetic, antispasmodic, antioxidant, antibacterial, and immunomodulatory activities, have been documented (Sajed et al. 2013). However, studies on the endophytic microorganisms inhabiting Z. multiflora are limited (Mohammadi et al. 2016).

Monosporascus cannonballus Pollack & Uecker (Ascomycota, Sordariomycetes, Diatrypaceae) is one of the most important phytopathogenic fungi causing root rot and vine decline disease in muskmelon. It causes sudden wilt and collapse of the plant at the fruiting stage, which may result in total yield loss (Martyn and Miller 1996). The fungus also infects pumpkin, cucumber, courgette, and watermelon plants (Mertely et al. 1993). The control of M. cannonballus in melon and other cucurbit crops is difficult because of the pathogen’s soil-borne nature. Earlier reports indicated that arbuscular mycorrhizal fungi (AMF) (Aleandri et al. 2015), hypovirulent isolates of M. cannonballus (Batten et al. 2000), Trichoderma spp. (Zhang et al. 1999), and antagonistic rhizobacteria (Al-Daghari et al. 2020) are effective agents for the reduction of M. cannonballus-induced root rot and vine decline of melon. In addition, it is well established that many endophytic fungi isolated from medicinal plants possess antimicrobial activity against phytopathogenic fungi (Jia et al. 2016). The objective of this study was to investigate the presence of endophytic fungi in Z. multiflora and to study theirs in vitro antagonistic activity against M. cannonballus.

Z. multiflora plants (accession number 201100114) were obtained from Oman Botanic Garden, Al-Khoud, Sultanate of Oman. The plants were healthy, showing no apparent symptoms of any disease or pest infestation. A virulent isolate of M. cannonballus (ID14367), obtained from the roots of a melon plant showing root rot and vine decline (Al-Rawahi et al. 2018) was used in this study. The culture was maintained on potato dextrose agar (PDA) medium (Oxoid Ltd., Basingstoke, UK).

To isolate endophytic fungi, Z. multiflora plants were washed in tap water to remove adhering soil particles. The leaves were separated, cut into small pieces, and surface-sterilized by washing in 70% (v/v) ethanol for 1 min and then in 1% (v/v) sodium hypochlorite for 1 min. The plant tissues were then washed 3–4 times with sterilized distilled water. The leaf tissue pieces were further cut into small pieces (0.2–0.5 cm in length) using a sterile scalpel and placed on PDA medium. The plates were incubated at 25 ± 2°C for 7–10 days, and pure cultures of the endophytic fungi were obtained (Lu et al. 2012).

DNA was extracted from the mycelia for molecular identification of endophytic fungi according to the method described by Liu et al. (2000). PCR amplification of the Internal Transcribed Spacer (ITS) regions of the fungal rDNA was performed using the primers ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) and ITS5 (5’-GGAAGTAAAAGTCGTAACAAGG-3’) as described by Halo et al. (2018). The PCR products of the expected sizes were sequenced at Macrogen, Seoul, Korea. The sequences were subjected to BLAST searches using the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov).

A dual culture technique was used to test the in vitro antagonistic effect of the endophytic fungi against M. cannonballus. A mycelial plug (7-mm diameter) was excised from the fungal endophyte colonies and placed on one side of a PDA plate (90-mm diameter) about 1 cm away from the edge. On the same plate, a 7-mm diameter disc of M. cannonballus was placed on the opposite side at 1 cm distance from the edge. The Petri plates inoculated with M. cannonballus alone were used as control. Four Petri plates per treatment were used. The Petri plates were incubated at 25 ± 2°C, and the radial growth of M. cannonballus was measured after 5–7 days of incubation. The mycelial growth inhibition was calculated using the following formula:

% inhibition=[1(T/C)]×100

where C – radial growth of M. cannonballus in the control plate and T – radial growth of M. cannonballus in the dual culture plate (Toghueo et al. 2016).

To investigate the antagonistic effects of the endophytic fungi on the morphology of M. cannonballus hyphae, the five-mm agar plug samples of M. cannonballus were excised from the colony edges of inhibition zone in the dual culture plate. The samples for scanning electron microscopy were prepared according to the method reported by Goldstein et al. (2003) and observed with a JEOL (Model: JSM-7800F) scanning electron microscope. The culture of M. cannonballus grown in the absence of endophytic fungi served as control.

To perform the electrolyte leakage assay, the endophytic fungi were cultured in 200 ml of Czapek Dox broth (static) in 500 ml conical flasks at room temperature (25 ± 2°C) for 14 days, and the culture filtrates were obtained by filtering through Whatman No. 1 filter paper. Five hundred mg of M. cannonballus mycelium were added to 20 ml of culture filtrate in a glass vial. The conductivity of the suspension was measured at 0, 1, and 3 h after incubation by using a conductivity meter (Halo et al. 2018). There were three replicates per treatment and control.

Data from the in vitro growth inhibition and the electrolyte leakage assays were statistically analyzed using general linear model ANOVA using Minitab Statistical Software version 17 (Minitab Inc., State College, USA). When ANOVA revealed significant differences between treatments, means were separated using Tukey’s studentized range test at p ≤ 0.05. Arc sine transformation of data on % mycelial growth inhibition was done prior to analysis.

A total of five morphologically distinct fungal endophytes were obtained from the leaves of Z. multiflora. Based on the rDNA ITS sequence analysis, these endophytic fungal (Ascomycota, Sordariomycetes) isolates were identified as Nigrospora sphaerica (Amphisphaeriales, Apiosporaceae) (E1 and E6), Subramaniula cristata (Sordariales, Chaetomiaceae) (E7) and Polycephalomyces sinensis (Hypocreales, Ophiocordycipitaceae) (E8 and E10). The sequences were deposited in the GenBank database (http://www.ncbi.nlm.nih.gov/genbank/) under the accession numbers MH028052, MH028054, MH028055, MH028056, and MH028058. P. sinensis is an important medicinal fungus. Numerous pharmacological activities of P. sinensis including immunomodulatory, anti-estrogenicity and antitumor activities have been documented (Wang et al. 2012). N. sphaerica has been reported as an endophyte (Wang et al. 2017) as well as a pathogen in a few plant species (Wright et al. 2008; Liu et al. 2016). However, Z. multiflora plants colonized with these endophytic fungi were healthy and did not show any observable disease symptoms.

The in vitro dual culture antagonism assay showed that all the five endophytic fungi inhibited the mycelial growth of M. cannonballus. N. sphaerica E1 was the most effective (81.7%), followed by P. sinensis E8 (80.6%), P. sinensis E10 (75.8%) and N. sphaerica E6 (66.1%). S. cristata E7 was the least effective, which recorded 38.7% inhibition (Table I, Fig. 1). Further, scanning electron microscopic observations of the hyphae of M. cannonballus from the dual culture assay plates at the edge of the inhibition zone revealed morphological abnormalities such as disintegration, shrinkage, and loss of turgidity. Scanning electron micrograph of M. cannonballus after co-cultivation with the endophytic fungus N. sphaerica E1 is shown in Fig. 2. These findings corroborate with those of Hajlaoui et al. (1992) who reported plasmolysis of Sphaerotheca pannosa var. rosae mycelium due to the antagonistic effect of Sporothrix flocculosa. Halo et al. (2018) reported shrinkage of Pythium aphanidermatum hyphae due to the antagonistic activity of Aspergillus terreus. The shrinkage of M. cannonballus hyphae in the present study suggests a possible leakage of cytoplasmic contents (Garg et al. 2010). The loss of the turgidity of M. cannonballus hyphae indicates alterations in the permeability of the cell membrane (Halo et al. 2018). Several reports indicate the production of antimicrobial substances by endophytic fungi (Zhao et al. 2012; Homthong et al. 2016). Kim et al. (2001) demonstrated that phomalactone, a compound produced by N. sphaerica restricted the mycelial growth and germination of sporangium and zoospore of Phytophthora infestans and decreased the incidence of late blight in tomato. Zhao et al. (2012) characterized four secondary antifungal metabolites viz., dechlorogriseofulvin, griseofulvin, mullein, and 8-dihydroramulosin from the liquid cultures of the endophytic fungus Nigrospora sp. isolated from roots of the medicinal plant, Moringa oleifera. Homthong et al. (2016) reported the production of chitinase by Paecilomyces (Polycephalomyces) sp. The inhibitory effect of endophytic fungi on the hyphae of M. cannonballus in this study might be due to the production of antifungal metabolites.

Table I

Percentage inhibition of mycelial growth of M. cannonballus by endophytic fungi isolated from Zataria multiflora in dual cultures on PDA.

10.33073_pjm-2020-029-tbl1.jpg
Fig. 1.

In vitro growth inhibition of Monosporascus cannonballus after dual cultivation with several endophytic fungi from Zataria multiflora.

a) M. cannonballus (Mc) alone; b) M. cannonballus + N. sphaerica E1; c) M. cannonballus + N. sphaerica E6; d) M. cannonballus + S. cristata E7; e) M. cannonballus + Paecilomyces sinensis E8; f) M. cannonballus + P. sinensis E10

10.33073_pjm-2020-029-f001.jpg
Fig. 2.

Scanning electron micrograph showing morphological changes in the hyphae of Monosporascus cannonballus at the edge of the inhibition zone after co-cultivation with Nigrospora sphaerica E1 in PDA plates

a) Hyphae of M. cannonballus in the control; b) Hyphae of M. cannonballus after co-cultivation with N. sphaerica E1.

10.33073_pjm-2020-029-f002.jpg

Several reports indicate that leakage of electrolytes is an indicator of cell membrane damage in fungi (Manhas and Kaur 2016; Halo et al. 2018). The present study observed that the culture filtrates of endophytic fungi induced electrolyte leakage from the mycelium of M. cannonballus as assessed by increased conductivity of mycelial suspension upon treatment with the culture filtrates of endophytic fungi (Table II). The maximum release of electrolytes was observed with N. sphaerica E1, followed by N. sphaerica E6, P. sinensis E10, S. cristata E7, and P. sinensis E8. The results suggest the production of antifungal metabolites as one of the possible mechanisms of action of these fungal endophytes on M. cannonballus.

Table II

Electrolyte leakage induced by culture filtrates of endophytic fungi from the mycelium of M. cannonballus.

10.33073_pjm-2020-029-tbl2.jpg

To our knowledge, this study is the first to report in vitro inhibitory activity of fungal endophytes isolated from Z. multiflora against M. cannonballus. Further studies are needed to evaluate the potential of these fungal endophytes in controlling root rot and vine decline disease of melon, assess their endophytic movement in melon plant, and to determine the mode of action of these fungal endophytes on M. cannonballus.

ORCID

Velazhahan Rethinasamy https://orcid.org/0000-0002-9263-4371

Acknowledgments

This work was supported in part by SQU through the projects IG/AGR/CROP/18/01, IG/AGR/CROP/16/03, and CR/AGR/CROP/19/01. We thank Dr. Khalid Al-Farsi and Dr. Annette Patzelt, Oman Botanic Garden for providing Zataria multiflora and Dr. Myo Tay Zar Myint, Department of Physics, College of Science, SQU for his help with scanning electron microscopy.

Conflict of interest

The authors do not report any financial or personal connections with other persons or organizations, which might negatively affect the contents of this publication and/or claim authorship rights to this publication.

References


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FIGURES & TABLES

Fig. 1.

In vitro growth inhibition of Monosporascus cannonballus after dual cultivation with several endophytic fungi from Zataria multiflora.

a) M. cannonballus (Mc) alone; b) M. cannonballus + N. sphaerica E1; c) M. cannonballus + N. sphaerica E6; d) M. cannonballus + S. cristata E7; e) M. cannonballus + Paecilomyces sinensis E8; f) M. cannonballus + P. sinensis E10

Full Size   |   Slide (.pptx)

Fig. 2.

Scanning electron micrograph showing morphological changes in the hyphae of Monosporascus cannonballus at the edge of the inhibition zone after co-cultivation with Nigrospora sphaerica E1 in PDA plates

a) Hyphae of M. cannonballus in the control; b) Hyphae of M. cannonballus after co-cultivation with N. sphaerica E1.

Full Size   |   Slide (.pptx)

REFERENCES

  1. Al-Daghari DSS, Al-Sadi AM, Janke R, Al-Mahmooli IH, Velazhahan R. Potential of indigenous antagonistic rhizobacteria in the biological control of Monosporascus root rot and vine decline disease of muskmelon. Acta Agric Scand B-S P. 2020;70(5):371–380. https://doi.org/10.1080/09064710.2020.1748703
  2. Aleandri MP, Martignoni D, Reda R, Chilosi G. Effects of preconditioning through mycorrhizal inoculation on the control of melon root rot and vine decline caused by Monosporascus cannonballus. J Phytopathol. 2015 Dec;163(11–12):898–907. https://doi.org/10.1111/jph.12389
    [CROSSREF]
  3. Al-Rawahi AY, Al-Mahmooli IH, Al-Sadi AM, Al-Sabahi JN, Velazhahan R. Toxin production by melon root rot fungus, Monosporascus cannonballus. Australas Plant Pathol. 2018 Sep; 47(5): 543–546. https://doi.org/10.1007/s13313-018-0589-1
    [CROSSREF]
  4. Bacon CW, White JF. Microbial Endophytes. New York (USA): Marcel Dekker Inc; 2000.
    [CROSSREF]
  5. Batten JS, Scholthof KBG, Lovic BR, Miller ME, Martyn RD. Potential for biocontrol of Monosporascus root rot/vine decline under greenhouse conditions using hypovirulent isolates of Monosporascus cannonballus. Eur J Plant Pathol. 2000;106(7): 639–649. https://doi.org/10.1023/A:1008703410411
    [CROSSREF]
  6. Garg H, Li H, Sivasithamparam K, Kuo J, Barbetti MJ. The infection processes of Sclerotinia sclerotiorum in cotyledon tissue of a resistant and a susceptible genotype of Brassica napus. Ann Bot (Lond). 2010 Dec;106(6):897–908. https://doi.org/10.1093/aob/mcq196
    [CROSSREF]
  7. Goldstein J, Newbury DE, Joy DC, Lyman CE, Echlin P, Lifshin E, Sawyer L, Michael JR. Scanning Electron Microscopy and X-Ray Microanalysis, 3rd Edition. Berlin (Germany): Springer; 2003.
    [CROSSREF]
  8. Hajlaoui MR, Benhamou N, Belanger NR. Cytochemical study of the antagonistic activity of Sporothrix flocculosa on rose powdery mildew, Sphaerotheca pannosa var. rosae. Phytopathology. 1992; 82(5):583–589. https://doi.org/10.1094/Phyto-82-583
    [CROSSREF]
  9. Halo BA, Al-Yahyai RA, Al-Sadi AM. Aspergillus terreus inhibits growth and induces morphological abnormalities in Pythium aphanidermatum and suppresses Pythium-induced damping-off of cucumber. Front Microbiol. 2018 Feb 1;9:95. https://doi.org/10.3389/fmicb.2018.00095
    [PUBMED] [CROSSREF]
  10. Homthong M, Kubera A, Srihuttagum M, Hongtrakul V. Isolation and characterization of chitinase from soil fungi, Paecilomyces sp. Agric Nat Resour (Bangk). 2016 Jul;50(4):232–242. https://doi.org/10.1016/j.anres.2015.09.005
  11. Jia M, Chen L, Xin HL, Zheng CJ, Rahman K, Han T, Qin LP. A friendly relationship between endophytic fungi and medicinal plants: a systematic review. Front Microbiol. 2016 Jun 09;7:906. https://doi.org/10.3389/fmicb.2016.00906
    [PUBMED]
  12. Kim JC, Choi GJ, Park JH, Kim HT, Cho KY. Activity against plant pathogenic fungi of phomalactone isolated from Nigrospora sphaerica. Pest Manag Sci. 2001 Jun;57(6):554–559. https://doi.org/10.1002/ps.318
    [PUBMED] [CROSSREF]
  13. Liu D, Coloe S, Baird R, Pederson J. Rapid mini-preparation of fungal DNA for PCR. J Clin Microbiol. 2000 Jan;38(1):471.
    [PUBMED]
  14. Liu YJ, Tang Q, Fang L. First report of Nigrospora sphaerica causing leaf blight on Camellia sinensis in China. Plant Dis. 2016 Jan;100(1):221. https://doi.org/10.1094/PDIS-04-15-0493-PDN
    [CROSSREF]
  15. Lu Y, Chen C, Chen H, Zhang J, Chen W. Isolation and identification of endophytic fungi from Actinidia macrosperma and investigation of their bioactivities. Evid Based Complementary Altern Med. 2012;382742. https://doi.org/10.1155/2012/382742
  16. Manhas RK, Kaur T. Biocontrol potential of Streptomyces hydrogenans strain DH16 toward Alternaria brassicicola to control damping off and black leaf spot of Raphanus sativus. Front Plant Sci. 2016 Dec 16;7:1869. https://doi.org/10.3389/fpls.2016.01869
    [PUBMED] [CROSSREF]
  17. Martyn RD, Miller ME. Monosporascus root rot and vine decline: an emerging disease of melons worldwide. Plant Dis. 1996;80(7):716–725. https://doi.org/10.1094/PD-80-0716
    [CROSSREF]
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