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Fungal interaction between Trichoderma spp. and Pleurotus ostreatus on the enriched solid media with licorice Glycyrrhiza glabra root extract

by Sajid Al-Saeedi (Author) Behar Al-Ani (Author) Mustafa Nadhim Owaid (Author)

Academic Paper 2017 6 Pages

Biology - Miscellaneous

Excerpt

ABSTRACT

The aim of this study is to investigate the antifungal activity of mycelia of Pleurotus ostreatus (white oyster mush­room) and licorice (Glycyrrhiza glabra) root extract against three undesirable fungi. They are Trichoderma spp., Trichoderma harzianum I and Trichoderma harzianum II which was tested on PSA (potato sucrose agar) medium enriched with licorice (Glycyrrhiza glabra) root extract (PSA-G media) using three concentrations (0.05, 0.10 and 0.20 g/L) in alone and dual cultures. Trichoderma spp. showed less mycelial growth of 8.75, 9.17 and 9.50 mm/day on PSA-G0.05, PSA-G0.1 and PSA-G0.2 respectively compared with 10.25 mm/day on fresh PSA (con­trol) in dual culture. The best mycelial growth inhibition was recorded on PSA-G0.2 (14.97%) by T. harzianum II in alone culture opposite 63.72% in dual ones. The lower mycelial growth rate of T. harzianum I was 17.75 mm/day on PSA-G0.1 (0.10 g/L). In dual culture, overgrowth time of T. harzianum I had 5 days compared as approx. 6 days in alone culture. Generally, when the concentration of licorice extract increased, the mycelial growth rate of the un­desirable fungi decreased. Also, all PSA-G media, especially PSA-G0.2, indicated low growth averages compared with the control (fresh PSA) against the pathogen while this concentration encourages growth of oyster mush­room. Also, this concentration reduced the density of sporulation of green molds; therefore, this concentration can be applied to reduce influence this pathogen in cultivation farm.

© 2017 Ecological Society of China. Published by Elsevier B.V. All rights reserved.

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1. Introduction

The fungus Pleurotus ostreatus, oyster mushroom, is one of the popu­lar macro-fungi which belong to higher Basidiomycetes. It is considered good food and drug sources as antioxidant, antibacterial and antifungal bio-factors [1]. In Iraq, Pleurotus ostreatus can be produced and grew on different cellulosic substrate sources such as wheat straw, cardboard wastes [2] and date-palm residues successfully [3].

In commercial mushroom farms, Trichoderma harzianum, T. longibrachiatum, and T. atroviride cause green molds [4]. T. harzianum, the most dominant contaminant, is leading to green mold disease and causing a high loss in Agaricus bisporus, P. ostreatus and Lentinula edodes production [5]. Also, Trichoderma spp. is a common contaminant of spawn, compost, and wood in mushroom cultivation farm [6]. Generally, in dual culture test, T. harzianum was antagonistic to Pleurotus spp. [7]. Whereas, mycelia of oyster mushrooms can be cover the T. harzianum colonies when supporting the medium using oils of tea tree Melaleuca alternifolica [6].

T. harzianum overgrew the colony of P. ostreatus rapidly due to me­tabolites of T. harzianum which reduced P. ostreatus growth in dual cul­tures. In contrast, metabolites of P. ostreatus were not stimulated growth of T. harzianum [8]. Also, T. harzianum has a highly aggressive toward A. bisporus, P. ostreatus and L. edodes that regarding contamination prob­lem which leads to a decrease in the gross production and the financial loss for producers [5]. From the incubation temperature profiles of the pathogen and oyster mushroom, no range was found that would allow optimal growth of the mushrooms without mold contamination [9].

Licorice or liquorice plant, Glycyrrhiza sp., is one of the medicinal plants from 4000 years ago, which used as a drug in different fields. It belongs to the family Fabaceae [10]. The primary active constituent of Glycyrrhiza sp. is Glycyrrhizin known as glycyrrhetinic acid or glycyrrhizic acid. It is fifty times sweeter than sugar (sucrose) with con­centration 6-14% [11]. From other side, Glycyrrhiza sp. is possible to se­crete flavonoids, coumarins [12], triterpenoids (Glycyrrhizin and volatiles) [13], glabrol (isoflavonoids) [14], tannins [15], licoricone, kumatakenin, chalcones and phytosterols, [11]. Many studies made up toward using licorice extract to encourage the growth of some plants and mushrooms because of naturally occurring compounds [16- ^.Ac­cordingly, Friis-Moller et al. [19] tested licochalcone A from licorice root powder toward anti-mycobacterial activity. Musa et al. [15] referred to Glycyrrhizin (glycyrrhizic) have an inhibitory effect toward microbes.

http://dx.doi.org/10.1016/j.chnaes.2017.08.001

1872-2032/© 2017 Ecological Society of China. Published by Elsevier B.V. All rights reserved.

B.M. Al-Ani etal. / Acta Ecologica Sinica xxx (2017) xxx-xxx

Also, using licorice extract spray on pomegranate fruits lead to deter­mine findings of microbes [20].

Strategies of possible management included the application of chem­ical treatments like adjustment of pH [21], disinfectant, pasteurization and biological control using antagonistic bacteria or using resistant mushroom varieties [22]. Thus this work dealt a new strategy (using Glycyrrhiza glabra licorice root extract) to inhibit and/or reduce the growth of those fungal pathogens and encourage the growth of P. ostreatus oyster mushroom at the same time in alone and dual cultures.

2. Materials and methods

2.1. Fungal strains

Three varieties of genus Trichoderma viz.; Trichoderma spp. obtained from Department of Horticulture, College of Agriculture; T. harzianum I and T. harzianum II obtained from fungi and plant pathology Lab. Depart­ment of Biology in University of Anbar, Iraq. White oyster mushroom Pleurotus ostreatus obtained from MushroomBox, UK which used for the mycelial interaction test.

2.2. Licorice root extract preparation

Licorice (Glycyrrhiza glabra) extract was prepared using topical lico­rice root powder with distilled water at ratio (1:10) (w/v), left for 24 h in shaker incubator at 25 °C. It was filtrated using Whatman No. 1, centri­fuged at 3000 cycle/min for 10 min and micro-filtrated using Millipore 0.22 p [17]. The treatments were three concentrations of G. glabra 0.05, 0.10 and 0.2 g/L (as symbolized by PSA-G0.05, PSA-G0.1, and PSA-G0.2 respectively) which used in this test in comparison with the control (PSA).

2.3. PSA medium supplemented with licorice extract

Potato sucrose agar (PSA) medium was prepared as mentioned by Owaid [17]. The prepared PSA medium was autoclaved at 121 °C for 25 min and then poured into 85 mm Petri dishes. Before solidity PSA, three concentrations of the plant extract were added to yield PSA- G0.05 (0.05 g/L), PSA-G0.1 (0.10 g/L) and PSA-G0.2 (0.20 g/L) [17]. Fresh PSA was considered as control.

2.4. The mycelial interaction between Pleurotus ostreatus and Trichoderma isolates

The mycelium of Pleurotus ostreatus (white) was tested for antifungal activity against three pathogenic fungi for mushroom cultivation farm viz.; Trichoderma spp., T. harzianum I and T. harzianum II on PSA medium enriched with licorice extract in three concentrations using dual culture method. Culture disks measuring 5 mm were made in the Petri dishes for each pathogenic fungus. A culture plug of seven-day-old pathogenic fungi was placed 3 cm away from the mushroom culture plug. The plates were then incubated for seven days at 25 ± 2 °C. Plates were checked and inhibition zone was measured and recorded in comparing with con­trol plates [7]. The number of days taken to overgrow the pathogens was recorded.

2.5. Percent inhibition of the mycelial growth

The seven-day-old cultures of three pathogenic fungi were placed in the center of plates and incubated at 25 °C for 2 days to detect inhibitory activity by measuring the mycelial growth rate (MGR) on PSA-G media in case dual (MGRDua]) or alone (MGRpsa_g) cultures and the radial growth on fresh PSA plate as control (MGRPSA). The experiment was car­ried out in three replicates. The percent inhibition was calculated using

Table 1

Mycelial growth rate of Trichoderma spp. on PSA-G media after 2 days in alone culture (mm/day).

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PSA: fresh potato sucrose agar medium, PSA-G0.05: PSA medium with licorice (G. glabra) extract at 0.05 g/L, PSA-G0.1: PSA medium with licorice extract at 0.10 g/L, PSA-G0.2: PSA medium with licorice extract at 0.20 g/L.

these equations:

Inhibition%(in dual culture) = [(MGRPSA-MGRDual)/MGRPSA] x 100. Inhibition%(in alone culture) = [(MGRPSA-MGRreA_G)/MGRPSA] x 100.

2.6. Sporulation of green molds

There are three levels ofsporulation density, which were observed on plates in case of alone and dual cultures as mentioned by [23]:

( + + + ): growth and sporulation similar to those of the wild-type strain

( + + ): reduced growth or/and sporulation (+): highly reduced growth or/and sporulation (_): no sporulation formation.

2.7. Statistical analysis

Statistical significance was determined by using CRD at two-way analysis of variance (ANOVA) by implementing GenStat Discovery Edi­tion computer program version 7 DE3 (VSN International Ltd., UK). Sig­nificant differences at p < 0.05 were considered. All the experiments were done in three replicates.

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Fig. 1. Pattern of radial mycelial growth of Trichoderma spp. on PSA media supplemented with licorice rootextract. Legend: PSA: fresh potato sucrose agarmedium, PSA-G0.05: PSA medium with licorice (G. glabra) extract at 0.05 g/L, PSA-G0.1: PSA medium with licorice extract at 0.10 g/L, PSA-G0.2: PSA medium with licorice extract at 0.20 g/L.

Table 2

Mycelial growth rate of Trichoderma spp. on PSA-G media after 2 days in dual culture (mm/day).

Abbildung in dieser Leseprobe nicht enthalten

PSA: fresh potato sucrose agar medium, PSA-G0.05: PSAmediumwith licorice (G. glabra) extract at 0.05 g/L, PSA-G0.1: PSA medium with licorice extract at 0.10 g/L, PSA-G0.2: PSA medium with licorice extract at 0.20 g/L.

Table 3

Percent inhibition of Trichoderma spp. on PSA-G media after 2 days in alone culture.

Abbildung in dieser Leseprobe nicht enthalten

PSA: fresh potato sucrose agar, PSA-G0.05: PSA medium enriched with licorice ( G. glabra) extract at 0.05 g/L, PSA-G0.1: PSA medium enriched with licorice extract at 0.10 g/L, PSA- G0.2: PSA medium enriched with licorice extract at 0.20 g/L.

3. Results

3.1. Radial mycelial growth of Trichoderma spp.

In alone culture test, mycelial growth rate (MGR) of Trichoderma spp. on PSA enriched with extract of licorice (G. glabra) root in three concen­trations (0.05, 0.10 and 0.2) g/L are presented in Table 1. The best MGR showed on fresh PSA medium for T. harzianum II and Trichoderma spp. at averages 36.75 and 36.50 mm/day respectively, then declined signifi­cantly (p < 0.05) to average 34.75 and 34.50 mm/day for Trichoderma spp. and T. harzianum II on PSA-G0.05 medium (0.05 g/L) respectively. While the lower MGR was 17.75 mm/day recorded on PSA-G0.1 (0.10 g/L), whereas, PSA-G0.2 recorded lower MGR for T. harzianum II and Trichoderma spp. of 31.25 and 33.50 mm/day respectively. Generally, all PSA-G media indicated low growth averages compared to the control (Table 1). Fig. 1 showed radial mycelial growth of Trichoderma spp.

In dual culture test (Table 2), mycelial interaction of Pleurotus ostreatus (white) antagonistic Trichoderma spp. was applied on PSA enriched with licorice extract. Generally, according to varieties of Trichoderma, bigger MGR was recorded 14.31 and 14.22 mm/day for Trichoderma spp. and T. harzianum II respectively, whereas, T. harzianum

1 had been less MGR at average 9.41 mm/day significantly (p < 0.05).

In another meaning, T. harzianum II had lower MGR 13.33 mm/day on PSA-G0.2 compared with 15.50 mm/day on fresh PSA (control). The sec­ond, Trichoderma spp. recorded 13.67 mm/day on PSA-G0.1 compared with 14.83 mm/day on the control medium. The last, T. harzianum I showed the lowest mycelial growth 8.75, 9.17 and 9.50 mm/day on PSA-G0.05, PSA-G0.1 and PSA-G0.2 compared with 10.25 mm/day on control (fresh PSA) in dual culture with P. ostreatus.

3.2. Sensitivity of Trichoderma spp. isolates toward PSA-G media

In Fig. 2, strains of Trichoderma spp. differ in sensitivity toward Pleurotus ostreatus. Mycelial growth of T. harzianum I showed lower sen­sitivity in alone (5.5 mm/day) and dual cultures (5 mm/day) during

2 days. In alone cultures, T. harzianum II and Trichoderma spp. showed mycelial growth about 3 mm/day, but showed approx. 4 mm/day in dual cultures.

Abbildung in dieser Leseprobe nicht enthalten

Fig. 2. Mycelial growth rate of Trichoderma spp. after 2 days in general.

3.3. Inhibition percent of green molds using PSA-G media

It is evident from the Table 3, the significant differences (p < 0.05) were observed among the percent inhibition of various pathogenic fungi on serious concentrations of licorice root extract on solid medium (PSA-G medium). In significant (p < 0.05), the best mycelial growth inhi­bition was recorded on PSA-G0.2 (14.97%) by T. harzianum II, followed 14.46% and 12.93% by T. harzianum I and T. harzianum II on PSA-G0.1 me­dium respectively, then reached to 10.84% on PSA-G0.2 in case of T. harzianum I. The lower growth inhibition was 4.79% in case of Trichoderma spp. on PSA-G0.05. PSA-G0.1 considered the best treatment to inhibit Trichoderma spp. in alone culture while other varieties are given best treatment on PSA-G0.2.

The force of inhibition of mycelial P. ostreatus against mycelial patho­genic fungi viz., Trichoderma spp., T. harzianum I, and T. harzianum II (in dual culture) on PSA-G media after 2 days is presented in Table 4. Gener­ally, inhibition percent of Trichoderma spp. started from 59.18%-60.16% in case of licorice extract compared with 55.93% with fresh PSA (control). According to varieties of the fungi, T. harzianum I exhibited less sensitive compared with Trichoderma spp. and T. harzianum II with inhibition per­cent 54.62%, 60.78% and 61.28% respectively. The lower significant (p < 0.05) inhibition achieved by T. harzianum I on fresh PSA medium (50.60%), while the isolate T. harzianum II had more inhibition percent 63.72% on PSA-G0.2 medium.

3.4. Sporulation patterns of pathogens

The sporulation was differed according to verities of Trichoderma spp. which ultimately inhibited growth ofP. ostreatus. Generally, Trichoderma spp. showed higher sporulation density (Fig. 3C), followed by T. harzianum I (Fig. 3A), while the Fig. 3B showed less sporulation in the plate. From Fig. 3A and B, sporulation level decreased when licorice root extract increased in dual cultures. Whereas, in alone culture, same observation was seen but in higher density in each state, that demon­strated the action of P. ostreatus against these pathogens. Table 5 showed various degrees of sporulation, which decreased with dual cultures for T. harzianum I and Trichoderma spp. in the supplemented media with plant extract. T. harzianum II have not affected.

Legend: A: T. harziunam I, B: T. harziunam II, C: Trichoderma spp.

Table 4

Percent Inhibition of Trichoderma spp. on PSA-G media after 2 days in dual culture.

Abbildung in dieser Leseprobe nicht enthalten

LSD p < 0.05, fungi = 0.805, culture media = 0.929, fungi * media = 1.609. PSA: fresh po­tato sucrose agar, PSA-G0.05: PSA medium with licorice (G.glabra) extract at 0.05 g/L,PSA- G0.1: PSA medium with licorice extract at 0.10 g/L, PSA-G0.2: PSA medium with licorice extract at 0.20 g/L.

Table 5

Sporulation levels of Trichoderma spp.

Abbildung in dieser Leseprobe nicht enthalten

Table 6

Number of overgrowth days of Trichoderma spp. on PSA-G media in alone culture.

Abbildung in dieser Leseprobe nicht enthalten

PSA: fresh potato sucrose agar, PSA-G0.05: PSA medium with licorice (G. glabra) extractat 0.05 g/L, PSA-G0.1: PSA medium with licorice extract at 0.10 g/L, PSA-G0.2: PSA medium with licorice extract at 0.20 g/L.

Table 7

Number of overgrowth days of Trichoderma spp. on PSA-G media in dual culture.

Abbildung in dieser Leseprobe nicht enthalten

LSD p < 0.05, fungi = 0.28, culture media = 0.32, fungi * media = 0.56. PSA: fresh potato sucrose agar, PSA-G0.05: PSA medium with licorice (G. glabra) extract at 0.05 g/L, PSA- G0.1: PSA medium with licorice extract at 0.10 g/L, PSA-G0.2: PSA medium with licorice extract at 0.20 g/L.

3.5. Comparison of overgrowth of green molds in alone and dual culture tests on PSA-G media

In alone culture test, the longer time used up to overgrow isolates of Trichoderma spp. on PSA-G media was 6 days for T. harzianum I on PSA- G0.05 and PSA-G0.1 media significantly (p < 0.05), then decreased to 5 days by the same fungus on PSA-G0.2 and fresh PSA media as well. While other isolates T. harzianum II and Trichoderma spp. had only 3 days to overgrow on all PSA-G media as seen in Table 6.

In Table 7, in dual culture, overgrowth of T. harzianum I had 5 days compared as (approx. 6 days) in alone culture (Table 6). However, T. harzianum II and Trichoderma spp. had more time for overgrowth when P. ostreatus was found, approx. 4 days in dual culture in comparison within the alone culture (3 days) on licorice extract media at different concentrations which appeared in Fig. 4. T. harzianum was inhibited by all PSA-G media in dual culture but P. ostreatus could not overgrow in presence of the fungal pathogen (Fig. 3A, B, C).

growth of pathogenic fungi using their secondary metabolism by-prod­ucts like triterpenoides, polysaccharides, proteins and enzymes [1]. Also, the ability of oyster mushroom to produce metabolic materials such as enzymes which give it inhibition force toward decay cellular walls of pathogenic fungi [26]. The difference of growth rate of Trichoderma isolates was useful to know the contact time between the green molds and oyster mushroom in vitro according to their inheritance characteristics.

Finally, P. ostreatus showed disability to overgrow T. harzianum. This could be due to hyphae of this pathogen that causes cell wall lysis at the point of interaction with oyster mushroom in Petri dish [27].

Also, Trichoderma species secrete hydrolytic enzymes including chitinases, (3-glucanases and cellulases which are kind of mycotoxins and lyse the fungal cell walls and are thought to play a role in the mycoparasitic activity of this fungus [28]. But that may be inhibit its bio­chemical pathways when adding licorice extract in some concentrations which did not effect on mycelia of oyster mushroom. In recent studies, phenolic compounds showed the remarkable fungistatic effect on the Trichoderma spp. and inhibited the host mushrooms as well, but licorice extract may be changing role of phenolic compounds toward them [9].

Inclusion, in dual culture, oyster mushroom mycelium has been given inhibition act against pathogenic fungi when be found G. glabra extracts as supporting natural materials in culture media especially at concentra­tion 0.2 g/L in this work at least. Trichoderma initially produces a dense pure white mycelium which resembles mushroom mycelium; therefore, they are very difficult to distinguish. Mycelial mat on the casing layer gradually turns to a green color because of the heavy sporulation of the causal agent producing a characteristic symptom of the disease [28], but when using this extract will lead to reducing the density of sporula- tion/growth of the pathogen as shown in Fig. 3 and Table 6.

Abbildung in dieser Leseprobe nicht enthalten

The high concentration of licorice G. glabra extract exhibited inhibito­ry effect toward oyster mushroom mycelia in this current study because of that 1-5% sucrose favorable to growth Pleurotus ostreatus but not more [24]. In opposite, using this plant extract showed anti-mycotic activity especially against its competitors Trichoderma isolates that due to the phenolic compounds (flavonoids and coumarins) of this extract [12]. While, this plant extract supports growth of some edible mushrooms like Pleurotus ostreatus as mentioned by Abdulhadi [16] who used licorice extract to support productivity and medicinal value of P. ostreatus, there­fore; it can be used to encourage producing oyster mushroom vigorously when the pathogenic fungi are found.

From other side, secondary metabolism of P. ostreatus mycelia was important against pathogenic fungi [25] because of its ability to inhibit

Abbildung in dieser Leseprobe nicht enthalten

Fig. 4. Effect treatments of licorice extract on growth completion of Trichoderma spp. (days). Legend: PSA: fresh potato sucrose agar, PSA-G0.05: PSA medium enriched with licorice (G. glabra) extract at 0.05 g/L, PSA-G0.1: PSA medium enriched with licorice extract at 0.10 g/L, PSA-G0.2: PSA medium enriched with licorice extract at 0.20 g/L.

This work was supported by the Department of Biology, College of Science, University of Anbar, Iraq. We would like to express my sincere gratitude to the department and the university.

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[*]Corresponding author at: Department of Heet Education, General Directorate of Education in Anbar, Ministry of Education, Hit, Anbar 31007, Iraq.

E-mail address: mustafanowaid@gmail.com (M.N. Owaid).

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Year
2017
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fungal trichoderma pleurotus glycyrrhiza

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Title: Fungal interaction between Trichoderma spp. and Pleurotus ostreatus on the enriched solid media with licorice Glycyrrhiza glabra root extract