Accepted Manuscript

Antagonistic studies and hyphal interactions of the new antagonist Aspergillus

piperis against some phytopathogenic fungi in vitro in comparison with Trichoderma
harzianum

Samah A. El-Debaiky

PII: S0882-4010(17)30900-2
DOI: 10.1016/j.micpath.2017.10.041
Reference: YMPAT 2550

To appear in: Microbial Pathogenesis

Received Date: 24 July 2017

Revised Date: 18 October 2017
Accepted Date: 20 October 2017

Please cite this article as: El-Debaiky SA, Antagonistic studies and hyphal interactions of the new
antagonist Aspergillus piperis against some phytopathogenic fungi in vitro in comparison with
Trichoderma harzianum, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.10.041.

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 ACCEPTED MANUSCRIPT

Antagonistic Studies And Hyphal Interactions Of The

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New Antagonist Aspergillus piperis Against Some

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Phytopathogenic Fungi In Vitro In Comparison With

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Trichoderma harzianum

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Samah A. El-Debaiky
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Botany Department, Faculty of Science, Tanta University, Egypt.

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Tel.: 002-01005256803

E-mail address: Samaheldebaiky@science.tanta.edu.eg

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Abstract
The present study represents, for the first time, the detailed studies about the
hyphal interactions of Aspergillus piperis, as a new antagonist, against some isolated
plant pathogenic fungi (Alternaria alternata, Alternaria solani, Botrytis cinerea,
Sclerotium cepivorum and Sclerotinia sclerotiorum) in vitro. The bio-controlling
capability of A. piperis against the tested phytopathogens was tested using the dual

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culture method. This experiment revealed that A. piperis had antagonistic activity and
reduced the growth of the tested phytopathogens and grew over their mycelia in the

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paired plates. Also, several antagonistic mechanisms were recorded, in this study,
between A. piperis and the tested phytopathogens using the microscopic examination.

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The bio-controlling activity and the antagonistic mechanisms exhibited by the new
antagonist, A. piperis were compared with those obtained by the common antagonist,

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Trichoderma harzianum against the same phytopathogens. The obtained results showed
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that, A. piperis was more effective than T. harzianum in inhibiting all the tested species
in the dual culture plates. The best result was 81.85 % inhibition percentage against S.
sclerotiorum by A. piperis while, T. harzianum exhibits only 45.18%. Moreover, several
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antagonistic mechanisms and hyphal interactions were investigated among the hyphae
of both A.piperis and T. harzianum and the hyphae of the tested phytopathogens. These
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mechanisms were summarized as; mycoparasitism (coiling and penetration of the

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hyphae) and antibiosis in the form of lysis of the hyphal cells and spores, denaturation
and breaking of the hyphae. The indirect interaction (antibiosis) and the direct
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mycoparasitism were observed by A. piperis against all the tested phytopathogens, but it
attacked the hyphae and conidiophores of A. alternata by only the antibiosis interaction.
The microscopic examination revealed also that T. harzianum attacked the tested
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phytopathogens by both antibiosis and mycoparasitism except against A. solani which

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attacked only by mycoparasitism.

Keywords: Antagonistic activity; Aspergillus piperis; Trichoderma harzianum

Phytopathogens; Hyphal interactions; coiling.
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1. Introduction
Biological control of plant diseases is considered as successful alternative
method to control plant diseases where it is safe for human, animals and did not cause
environmental pollution (Barakat and Al-Masri, 2010). Chemical fungicides have
harmful effects on the environment especially in long term usage because they cause
pollution, leave harmful residues and may lead to the development of resistant strains of

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the pathogen with repeated use (Belete et al., 2015). Many plant pathogens are
managing by ecofriendly, potential and non-chemical antagonistic (bio-controlling)

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organisms (TEWARI and Bhanu, 2003)
Trichoderma spp. are soil fungi which are associated with the rhizosphere of

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many plants and are considered from the most important biological controlling agents
where they suppress the plant pathogens by different mechanisms, such as competition,

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mycoparasitism, antibiosis and induced systemic resistance (Belete et al., 2015).
Mycoparasitism has been proposed as the major antagonistic mechanism displayed by
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Trichoderma spp. (Kubicek et al., 2001). After host recognition, Trichoderma spp.
attaches to the host hyphae via coiling, and penetrate the cell wall by secreting cell wall-
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degrading enzymes (Viterbo et al., 2002).

There are a few studies describing Aspergillus from section Nigri as antagonists
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to plant pathogens. In earlier study, 10 fungal isolates from compost were antagonistic
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to F. oxysporum f. sp. melonis, where the best biological control activity was recorded
for two Aspergillus spp. section Nigri (Suárez-Estrella et al., 2007). Recently,
Aspergillus piperis was tested for its antagonistic activity and found to exhibit strong
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antifungal activity against the phytopathogen Pythium aphanidermatum. It secretes a

complex mixture of metabolites consisting of small molecules, including gluconic acid,
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citric acid and itaconic acid derivatives, but the most potent antifungal activity was
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associated with proteins resistant to heat and organic solvents (Jovičić-Petrović et al.,
2016).
There are several relationships and interactions among different microbial
communities. These interactions are numerous and range from synergistic and
mutualistic to antagonistic and parasitic (Duffy et al., 2003). The mechanisms of these
microbial interactions were summarized as competition, which occurs between
microorganisms when space or nutrients (i.e. carbon, nitrogen and iron) are limited.
Also, the inhibition of a microorganism by metabolic products such as antibiotics from
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the other microorganism is called antibiosis. Moreover, there are some microorganisms
which can attack and parasitize on the other parasites in a phenomenon called hyper-
parasitism which also called mycoparasitism when a fungus is parasitized by another
one (Arya and Perelló, 2010).
The mycoparasitism commonly occurs in nature by several methods which
lead to predation viz., coiling, penetration, branching, sporulation, resting body

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production, barrier formation and lysis (Fig.1)(Dubey and Dwivedi, 1986); (Dubey,
1993)

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In coiling (Fig.1A) the antagonist (a) recognizes its host hyphae (h), comes in
contact and coils around it then the host hypha loses its strength. If the antagonist can

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secret cell wall degrading enzymes it can penetrate the cell wall of host hyphae and
penetrated the lumen of the cells (Fig.1B). The degrading enzymes associated with this
process have been reported by (Elad et al., 1982) such as cellulase b-1, 3-glucanase,
chitinase, etc.
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Sometimes the host hypha develops a resistant barrier by accumulating the
cytoplasm to prevent the penetration of the antagonist inside the cell. (Fig.1C). Also,
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the antagonist can be branched and produce its spores (s) inside the host hypha
depending on nutrition (Fig.1D). After the host's nutrients deplete, the antagonist
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produces survival structures, such as chlamydospores (c) inside the host hypha (Fig.1E).
Finally, the host hypha has been lysed due to loss of nutrients (Fig.1F) (Dubey, 1993).
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In this field, few studies were interested in examining the details of hyphal
interactions and mechanisms among the hyphae of the antagonists and the plant
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pathogens. Where the main target of these studies was focused on protection of the plant
from the pathogenic microorganisms. Accordingly, the present study aimed to,
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investigate and examine the different antagonistic mechanisms and hyphal interactions
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between A. piperis as a new antagonist against some phytopathogenic fungi, which were
isolated from different diseased plants. Then a comparison between A. piperis and the
common antagonist T. harzianum was carried out using the dual culture tests and
microscopic examination.
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2. Materials and Methods

2.1 Source of T. harzianum and A. piperis:

Cultures of both antagonistic fungi; T. harzianum (AUMC No.11456) and A.

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piperis (AUMC No.9043) were purchased from the mycological center (AUMC),
Assuite University, Assuite, Egypt. Each of them was cultured and maintained on

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potato dextrose agar (PDA) plates and slants at 4°C ±2. All the experiments in this
study were performed using PDA which prepared according to (Moubasher, 1993).

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500 mg capsule of chloramphenicol antibiotic was added to the medium as
antibacterial agent for preventing the contamination.

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2.2 Isolation and identification of phytopathogenic fungi:
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The phytopathogenic fungi used in this study were isolated from different
diseased plants parts. Each of A. alternate, A. solani and B. cinerea were isolated from
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diseased tomato fruits while S. cepivorum was isolated from diseased onion bulbs
which collected from infected onion field, Tanta, Egypt. On the other hand, S.
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sclerotiorum was isolated from rotted strawberry fruit. In sterilized conditions, the
spores of A. alternata, A. solani and B. cinerea were transferred separately using
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sterilized needle, from spore full lesions on the spoiled fruit surface, on PDA plates
and incubated at 27°C ±2 for 5 days. Also, the sclerotia of S. cepivorum were picked
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from the infected onion bulb and surface sterilized by absolute ethanol for 10 min.
then washed twice by sterilized distilled water for 10 min. The sterilized sclerotia were
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transferred to PDA plates and incubated at 20°C ±2 for 10 days. Moreover, a small
piece of rotted strawberry fruit was cultured on PDA plates and incubated at 24°C±2
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for 20 days. The appeared sclerotia of S. sclerotiorum were picked and surface
sterilized by absolute ethanol for 10 min. then washed twice by sterilized distilled
water for 10 min and sub-cultured using new PDA plates. Pure cultures from all the
isolated fungi (Photo.1) were clearly examined and identified morphologically and
microscopically according to the identification keys cited in the identification books of
fungi such as (Gilman, 1957),(Ellis, 1971)& (Moubasher, 1993).
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2.3 Dual culture tests:

The ability of the new antagonist; A. piperis in bio-controlling and inhibition
of the tested phytopathogenic fungi was studied by a modified dual culture method
(Jamdar et al., 2013). Plates of PDA were inoculated separately with 10 mm disc of

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the tested phytopathogen, 10 mm from the edge of the plate. After that,10 mm disc of
each tested antagonistic fungus was placed separately in the same plates 60 mm far
from each of phytopathogen disc and plates were incubated at 27±2°C for 4 days.

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Control plates were inoculated separately with the phytopathogen only. Each
treatment was performed in triplicate. The mean diameter of the pathogens in dual

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cultures were compared to that in the control after four days for A. alternata, A. solani
and B. cinerea while for 10 days for S. sclerotiorum and S. cepivorum. The

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percentage growth inhibition (%) was calculated using the formula given by (Vincent,
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1947) and (Jayasinghe and Wijesundera, 1995):

I= (C – T)/C x 100
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Where I= percentage inhibition of fungal mycelial growth with respect to

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control, C= growth in control and T= growth in treatment.

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To evaluate the antagonistic activity of A. piperis, this experiment was repeated

using the common antagonist, T. harzianum against the same tested phytopathogens.
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2.4 Microscopic examination of the hyphal interactions:

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In dual culture plates, the contact regions between the antagonistic fungi and
each of the tested phytopathogenic fungi were investigated using binocular biological
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light microscope (Model: XSZ-107BN) at different magnification powers (40, 100,

400 and 1000 X). To observe the hyphal interactions, the slides were prepared by two
methods. The first method was done by taking small portion from the contact hyphal
region between the antagonist and the pathogen on a glass slide and mounted by
methylene blue. The second method was prepared by tacking a print from the contact
hyphal region using an adhesive tap then put it on a slide with drop of methylene blue.
The contact interactions between the fungal hyphae was examined using low power
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(40 and 100 X). The detailed hyphal parasitism, coiling, disintegration, denaturation
and penetration were observed using high power (400 X) and oil emersion (1000 X)
then photographed.

3. Results and discussion

3.1 Isolation and identification of phytopathogenic fungi:

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Pure cultures of the isolated tested phytopathogens were examined

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morphologically and microscopically. Different cultural and morphological characters
of the tested phytopathogens were represented in Table.1 and photo.1. A. alternata

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and A. solani are from dematiaceous fungi which characterized by their dark reverse in
bottom of the plates, however they are recognized from each other by culture
appearance and microscopic examination. Culture of A. alternata was characterized by

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its olivaceous black and grey color with olivaceous and brownish black color reverse
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(photo.1: A&B). Conidia were formed in long acropetally chains on simple and
straight conidiophores (photo.1C) which all are characterized by pale to mid
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olivaceous or golden brown color. Mature conidia of A. alternata are obclavate,

obpyriform, ovoid and ellipsoidal phragmospres or dictyospores with short conical or
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cylindrical peaks. Sometimes the conidial chains are branched (photo.1D). These
morphological and microscopical features of A. alternata is confirmed by (Nabahat et
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al., 2014) and (Basım et al., 2017) who illustrated the morphologicall characteristics of
different Alternaria species.
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Pure culture of A. solani was appeared light to dark brown when old, in the
middle of the colony, with rose color in the young growing edges. Its reverse was
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dark brown under the oldest part ranged to reddish brown and yellow under the edges
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(photo.1: E&F). Conidia were not recognized in the young cultures and scarce in the
oldest. These conidia are characterized by their long peaks (photo.1G). This
description of A. solani is agreed and supported by (Kumar et al., 2008).

The culture of B. cinerea was recognized by its brownish grey color with no
reverse except brownish black color under the oldest portion (photo.1: H&I). This
fungus was known as the anamorph of the ascomycetous Sclerotinia sp. so it formed
rigid black sclerotia at the edges of old cultures. The mycelium was composed of
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transparent grayish divided hypha. The dichotomously branched conidiophores were
ended with swollen cells which generated the conidia (photo.1J). Conidia are
ellipsoidal or ovoid, colorless to olivaceous grey and with smooth walls. These
features of B. cinerea were agreed those which described by (Khazaeli et al., 2012).

The white sterile mycelia of S. cepivorum were full of rigid, black, globose or
ellipsoidal sclerotia (Photo.1K). This fungus causing serious damages in onion crop

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where it was considered the causal agent of the white rot disease of onion bulbs. Its
resistant sclerotia stay in the soil and infecting the onion bulbs whenever present. This

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identification was supported by earlier study of (Georgy and Coley-Smith, 1982).

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The plates of S. sclerotiorum were recognized by the formation of large (3 x 4
mm to 3 x 6 mm), sub-globose to ellipsoidal, rigid, white at first then turned black
sclerotia. There were exudate droplets formed on the surface of the sclerotia

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(Photo.1L). The microscopic examination of the hypha revealed the presence of
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granules inside the hyphal cells (Photo.1M). All these features were specialized to S.
sclerotiorum from the other species of Sclerotinia. Other studies supported that
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identification such as (Ekins et al., 2005) (Kapatia et al., 2016).

3.2 Dual culture tests:

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The results of the present investigation indicated that A. piperis clearly

inhibited the growth of the tested phytopathogens. Also, the obtained inhibition
percentage by A. piperis were higher than those obtained by the common antagonist T.
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harzianum against the same phytopathogens. Table.2 and Photo.2 show the
antagonistic effects of both antagonists against the tested phytopathogenic fungi in the
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dual culture plates. T. harzianum and A. piperis, exhibited fast spreading growth over
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all the tested pathogens in the contact regions with notification that A. piperis was
faster than T. harzianum in spreading in the plates and restricted the pathogens in
small areas then began to overgrowth them. This growth behavior of A. piperis led to
higher inhibition percentage against the phytopathogens than T. harzianum.

The results indicated that, A. piperis was more effective against B. cinerea, S.
sclerotiorum and A. Alternata where the percentage of inhibition was above 80% (85,
81.85 and 81.38 % respectively) compared to the control (Photo. 2: A, C & E). In
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contrarily, T. harzianum had less inhibitory against the same pathogens where the
percentage of inhibition was 50, 45.18 and 61.58 % respectively (Photo. 2: B, D &
F). Table.2 and Photo.2 reveals that A. piperis and T. harzianum give medium effect
on S. cepivorum where the inhibition percentage was 73.55 and 74.88 respectively.
The two antagonists have approximately the same effect on S. ceivorum.
These results were in accordance with previous studies which represented that

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some species of Aspergillus niger group and some species of genus Trichoderma were
used as bio-controlling agents against some phytopathogenic fungi. A. niger was

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efficient in the suppression of rice sheath blight caused by Rhizoctonia solani
(Kandhari et al., 2000). A. piperis produced the highest inhibition percentage in P.

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aphanidermatum (81%) and little activity against B. cinerea and F. oxysporum (33%)
(Jovičić-Petrović et al., 2016). Moreover, the causal agent of black root rot of faba bean
(F. solani) was inhibited by some species of Trichoderma recording inhibition

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percentages from 33.9% to 67.0% (Belete et al., 2015). Also, Fusarium root rot
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caused by F. graminearum, foot rot disease on rice caused by F. verticilloides and
verticillium wilt were bio-controlled by some strains of Trichoderma spp.(Sempere
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and Santamarina, 2009)& (Foroutan, 2013).

3.3 Microscopic examination:

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3.3.1 Antagonistic effects of A. piperis and T. harzianum against A. alternata:

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Photo.3 shows the harmful effects on the somatic structures of A. alternata

after attacking its mycelium separately by both A. piperis and T. harzianum. The hyphae
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and conidiophores of A. alternata were denaturated when contacted with A. piperis

(Photo.3B). This may be occurring due to production of lytic enzymes and secondary
metabolites of A. piperis (Jovičić-Petrović et al., 2016). While, T. harzianum exhibited
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the mycoparasitism mechanism by its parasitic hyphae which were attached to the
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hyphae of A. alternata through coiling which finally led to cell denaturation and lysis
(Photo.3: C&D). Though, A. piperis attacked A. alternata by only the antibiosis
mechanism and, T. harzianum attacked by mycoparasitism and antibiosis, but, A. piperis
was more effective in inhibiting the pathogen growth than T. harzianum as shown
previously in the dual culture plates (Table.1 and Photo.2: E&F). My results using T.
harzianum was in agreement with (Sempere and Santamarina, 2007) who found the
hypha of T. harzianum grew parallel the hyphae of A. alternata then coiled around them
and formed appressoria. Subsequently T. harzianum penetrated the hyphal cell wall of
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A. alternate, by releasing chitinase enzyme, to use the cell contents as source of
nutrients. On the other hand, (Gveroska and Ziberoski, 2012) recorded deformations in
the hyphae of A. alternata as a result of the metabolites of T. harzianum after
microscopic examination of the contact region of the two fungi in dual culture.

3.3.2 Antagonistic effects of A. piperis and T. harzianum against A. solani:

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By examining the contact area between A. solani and A. piperis, an obvious
coiling by the hyphae of A. piperis around the hyphae of A. solani was noticed

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(Photo.4B). The hyphae of A. piperis also penetrated the hyphae of A. solani after
coiling then led to hyphal cells lysis because of enzymatic interactions (Photo.4: C&D).

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So, A. piperis uses the internal penetration and direct enzymatic lysis of the protoplasm
of the hyphal cells of A. solani. While, T. harzianum attacked externally by
mycoparasitism where it formed a network of coiled attacking hyphae around the

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hyphae of A. solani (Photo. 4: E&F). These mode of attacking needs firstly the lysis of
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hyphal cell walls by secretion of lytic enzymes then began to lyse the internal
components of the cells. This step may notify why T. harzianum delayed in its
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inhibition of the pathogen than A. piperis.

All these results clarify the higher inhibition percentage of A. piperis rather
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than T. harzianum against A. solani in the paired plates. There are no previous studies
illustrating the hyphal interactions among A. piperis and T. harzianum against A. solani
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but the previous studies interested by bio-controlling of the early blight disease caused
by A. solani using T. harzianum such as (Chowdappa et al., 2013),(Selim, 2015) and
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(Chohan et al., 2015).

3.3.3 Antagonistic effects of A. piperis and T. harzianum against B. cinerea:
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A.piperis and T. harzianum exhibited numerous antagonistic mechanisms and

hyphal interactions against the hyphae of B. cinerea. The hyphal interaction mechanisms
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performed by both antagonists were similar where they penetrated inside the pathogen
hyphal cells leading to their lysis (Photo.5: B, D, F & G). But, the hyphae of B. cinerea
appeared broken because of the presence of A. piperis, the phenomenon which was not
observed with T. harzianum (Photo.5C). The hyphae of T. harzianum coiled the
pathogenic hyphae and penetrated them by hocks (Photo.5E) which is a common
mechanism of T. harzianum and was found in the past by the researchers like (Cheng et
al., 2012) who illustrated that hyphae of T. harzianum attacking the hyphae of B. cinerea
by coiling the hyphae.
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Likewise, the results reported against the previously mentioned two
pathogens, A. piperis is still better than T. harzianum where it was more effective in
inhibiting B. cinerea (Table, 2). It might be due to the hyphal fission mechanism which
does not appeared in the presence of T. harzianum. Moreover, that the secondary
metabolites of A. piperis might be more toxic to the pathogen than those of T.
harzianum.

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3.3.4 Antagonistic effects of A. piperis and T. harzianum against S. cepivorum:

In this investigation, the hyphae of A. piperis attacked the hyphae of S. sepivorum

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by means of coiling which leading to disintegration of their cells (Photo.6: B&C). The
coiling process was also conducted by the hyphae of T. harzianum which surrounded the

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hyphae of S. cepivorum directly or by means of hocks which then led to fission in the
septa between the hyphal cells and lysis of the cells. (Photo.6: D-F). This mechanism

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was also reported by (Ahmed and Ahmed, 2015) who investigated that T. harzianum
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attacking the hyphae of S. cepivorum by mycoparasitism.
The two antagonists behaved the same mechanisms against S. cepivorum, this may
be illustrating the similarities in the inhibition percentage of them against this pathogen
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(Table, 2).
3.3.5 Antagonistic effects of A. piperis and T. harzianum against S.
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sclerotiorum:
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Photo.7 reveals that the hyphae of S. sclerotiorum were affected by the presence
of both antagonists. The harmful effect is appeared in lysis of some hyphal cell walls.
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Also, the hyphae of A. piperis sent haustoria penetrating the hyphae of S. sclerotiorum
for nourishment (Photo.7C). On the other hand, Photo.7: D&E shows that the hyphae of
T. harzianum directly penetrated the pathogenic hyphal cells. In addition (Inbar et al.,
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1996), found that dense coils of T. harzianum hyphae grew around the S. sclerotiorurm
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hyphae and partial degradation of the Sclerotinia cell wall were observed in later stages
of attacking. Also coiling by hyphae of T. harzianum on hyphae of S. sclerotiorum was
reported by (Abdullah et al., 2008) by producing hook-like and appressoria-like
structures, which enable the penetration of S. sclerotiorum hyphae. However, the
mechanism performed by A. piperis by sending haustoria to penetrate the hyphal cells of
the pathogen and absorbing its nutrients is more effective than the hyphal penetration by
T. harzianum in affecting the cell walls of the pathogenic hyphae. This was clear in
Table 2 where the inhibition percentage by A. piperis was twice that of T. harzianum.
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4. Conclusions
In the present study, some phytopathogenic fungi were isolated from diseased
tomato fruits, onion bulbs and rotted strawberry fruits then identified

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morphologically and microscopically. The activity of A. piperis as a new antagonist
against these phytopathogenic fungi was performed in vitro. It shows a great

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potentiality in inhibiting the mycelial growth of all the tested pathogens. The
present investigation is considered the first study examining in details the hyphal

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interaction characters of the new antagonist; A. piperis. Also, the obtained results
were compared with the common antagonistic fungus T. harzianum against the

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same tested phytopathogens. A. piperis exhibited better inhibition percentage
against the tested pathogens in the dual culture plates than T. harzianum. Several
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myco-parasitic mechanisms of both antagonists were explained microscopically;
coiling, cell denaturation and degradation, hyphal breaking, fission and penetration.
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The microscopic examination revealed that, A. piperis used mycoparasitism and

antibiosis interactions in inhibiting all the tested phytopathogens but it affected the
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hyphae and conidiophores of A. alternata by only antibiosis. Also, T. harzianum

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attacking the mycelia of the tested phytopathogens using the mycoparasitism and
antibiosis mechanisms except against A. solani where it was attacked by
mycoparasitism only. This study also cited that, the myco-parasitic activity, lytic
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effects and antibiosis of A. piperis is more effective than T. harzianum and faster in
inhibiting the tested phytopathogens. This may be due to the high toxicity of the
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secondary metabolites of A. piperis over those of T. harzianum. Finally, the present

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work is considered a starting point for further important studies about using the new
antagonist A. piperis in the bio-controlling of plant pathogens and in many fields of
sciences such as, plant pathology, antagonism, fungal toxins…….etc.
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5. References

Abdullah M.T., Ali N.Y., Suleman P. (2008) Biological control of Sclerotinia sclerotiorum (Lib.)

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de Bary with Trichoderma harzianum and Bacillus amyloliquefaciens. Crop
protection 27:1354-1359.
Ahmed H.A.M., Ahmed N.G. (2015) Management of white rot of onion using composts and

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Trichoderma harzianum. Current Life Sciences 1:63-69.
Arya A., Perelló A.E. (2010) Management of fungal plant pathogens Cabi.
Barakat R.M.B.M., Al-Masri M.I. (2010) Biological control of gray mold disease (Botrytis

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cinerea) on tomato and bean plants by using local isolates of Trichoderma
harzianum. 32 ‫ ا وم ا زرا‬:‫درا ت‬.
Basım E., Basım H., Abdulai M., Baki D., Öztürk N. (2017) Identification and characterization
of Alternaria alternata causing leaf spot of olive tree (Olea europaea) in Turkey. Crop

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Protection:79-88.
Belete E., Ayalew A., Ahmed S. (2015) Evaluation of local isolates of Trichoderma spp.
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against black root rot (Fusarium solani) on Faba bean. Journal of Plant Pathology &
Microbiology.
Cheng C.-H., Yang C.-A., Peng K.-C. (2012) Antagonism of Trichoderma harzianum ETS 323 on
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Table 1: Morphological characters of the tested phytopathogens:

PT
RI
U SC
AN
M
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT
Characters Culture Conidia Sclerotia

Color Reverse Shape and Peak Shape and Compact-

Phyto-
Color color ness
pathogen

A. alternata Olivaceous Olivaceous Obclavate, Short conical ‫ــــــــــ‬ ‫ــــــــــ‬

black and black and obpyriform, or cylindrical

PT
grey brownish ovoid or shape.
black ellipsoid. Pale to mid

RI
Pale to mid golden brown
olivaceous

SC
or golden
brown

U
A. solani Light to dark Dark Ellipsoidal. Cylendrical ‫ــــــــــ‬ ‫ــــــــــ‬
brown with brown to brown long peak
AN
rose at edges reddish
brown and
M

yellow

B. cinereal Grey None or Ovate or ‫ــــــــــ‬ Black. Rigid

D

brownish ellipsoidal. Found at the

TE

black Olivacieous edges of the old

under the grey. colonies
oldst part Smooth
EP

S. cepivorum White none ‫ــــــــــ‬ ‫ــــــــــ‬ Globose or Rigid

ellipsoidal.
C

Black
AC

S. sclerotiorum White none ‫ــــــــــ‬ ‫ــــــــــ‬ Sub-globose to Rigid

ellipsoidal with
exudate
droplets.
Black
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Table.2: Antagonistic effect of T. harzianum and A. piperis on the growth of the

tested phytopathogenic fungi:

PT
RI
SC
The antagonistic Percentage of Inhibition (%)

U
The phyto- fungi
T. harzianum A. piperis
pathogenic fungi
AN
A. alternata 61.58 81.38
A. Solani 54.33 71.85
M

B. cinerea 50 85
S. sclerotiorm 45.18 81.85
D

S. cepivorum 74.88 73.55

TE
C EP
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
Fig.1: Post-interaction events during mycoparasitism. A, coiling (a, antagonist; h, host
hypha); B, penetration; C, barrier formation (b) by host; D, branch formation and sporulation (s)
M

by antagonist; E, chlamydospore (c) formation; F, lysis of host hypha. (diagrammatic, after

Dubey and Dwivedi, 1986).
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT

PT
RI
SC
A B D

U
AN
M

E F
D
TE

G
C EP
AC

J
H I

K L M

Photo.1: Morphological and microscopic description of phytopathogens; A. alternata (A-D), A.

solani (E-G), B. cinerea (H-J), S. cepivorum (K) and S. sclerotiorum (L&M).
 ACCEPTED MANUSCRIPT

PT
RI
A B C D

U SC
AN
M

E F G H
D
TE
EP

I K
C
AC

Photo.2: Dual culture among A. piperis and T. harzianum against B. cinerea (A&B), S.
sclerotiorum (C&D), A. Alternata (E&F), S. cepivorum (G&H) and A. solani (I&K).
 ACCEPTED MANUSCRIPT

PT
Denaturated
Conidiophore
hyphal cells

RI
Intact hypha

SC
AA B Denaturated
conidiophore

U
AN
Hypha of T.
M

harzianum

Hypha of A.
D

alternata
C D D
TE
EP

Denaturated and
lysed hyphal cells
Photo.3: Antagonism and hyphal interactions between A. piperis and T. harzianum against A.
C

alternata. Control (A), with A. piperis (B) and with T. harzianum (C & D).
AC
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Hyphae of

PT
Intact hypha A. piperis
of A. solani

RI
Hypha of A.
solani
B

SC
A

U
AN
M

C
Lysed
D cell
D
TE

Hyphae of A. piperis
penetrated hypha of A. solani
EP

Hyphal net of
T. harzianum
surround
C

hypha of A.
solani
AC

E F

Attacking hyphae
of T. harzianum
Photo.4: Antagonism and hyphal interactions between A. piperis and T. harzianum against A.
solani. Control (A), with A. piperis (B-E) and with T. harzianum (F & G).
 ACCEPTED MANUSCRIPT

PT
RI
SC
A B C

U Denaturaed and
AN
lysed hyphal cells Spore of A. Breaking in the
piperis hypha
Hock
Hypha of A. piperis
M

Hypha of T. harzianum
Hypha of B. cinerea
D
TE
EP

D
C

E F
AC

Lysed cells

Hypha of B. cinerea Hypha of T. harzianum

Photo.5: Antagonism hyphal interactions between A. piperis and T. harzianum against B.

cinerea. Control (A), with A. piperis (B-D) and with T. harzianum (E-G).
 ACCEPTED MANUSCRIPT

PT
Lysed cell

RI
Coiling
A

SC
B
Sclerotium of S. cepivorum Hypha of S. cepivorum

U
AN
Hypha of S. cepivorum
M

Hypha of A. piperis

Lysed cell
D

C
TE

Hyphae of T. harzianum
EP

coiled Hypha of S.
cepivorum
C

Fission in hypha of S.
AC

cepivorum
E
D

Hocks from hyphae of T.

harzianum penetrating
Hypha of S. cepivorum
Fission in hypha of S.
cepivorum

F
 ACCEPTED MANUSCRIPT

PT
RI
SC
A

U
AN
Haustorium
M

Hypha of A. piperis

Lysis in hyphal
D

cell wall
TE

B
EP

D E
C

Lysis in hyphal cell wall

AC

Hyphae of T. harzianum
penetrating hypha of S.
sclerotiorum

Photo.7: Antagonism hyphal interactions between A. piperis and T. harzianum against S.

sclerotiorum. Control (A), with A. piperis (B&C) and with T. harzianum (D&E).
 ACCEPTED MANUSCRIPT
Research highlights:
1. Some phytopathogenic fungi were isolated from spoiled tomatoes, onions and
strawberry then identified morphologically.
2. The new antagonist, Aspergillus piperis, suppressed the growth of the isolated
phytopathogenic fungi.
3. Several hyphal interactions between the hyphae of A. piperis and the
phytopathogens were examined microscopically and summarized as;
mycoparasitism (coiling and penetration of the hyphae) and antibiosis in the

PT
form of lysis of the hyphal cells and spores, denaturation and breaking of the
hyphae.
4. All the antagonistic activity and hyphal interactions of A. piperis against the

RI
phytopathogens were compared to the common antagonist, Trichoderma
harzianum. The results indicated that A. piperis was more effective.

U SC
AN
M
D
TE
C EP
AC