International Journal of Environment, Agriculture and Biotechnology

Vol-10, Issue-5; Sep-Oct, 2025

Peer-Reviewed International Journal
Journal Home Page Available: https://ijeab.com/
Journal DOI: 10.22161/ijeab

Antifungal Activity of Endophytic Nigrospora Species

Isolated from Pluchea Plants against Some Fungal
Phytopathogens
Ahmed I. S. Ahmed and Hanan M. Zakaria

Plant Protection Department, Desert Research Center, Cairo, Egypt

Corresponding: [email protected]; [email protected]

Received: 05 Aug 2025; Received in revised form: 04 Sep 2025; Accepted: 08 Sep 2025; Available online: 15 Sep 2025
©2025 The Author(s). Published by Infogain Publication. This is an open-access article under the CC BY license
(https://creativecommons.org/licenses/by/4.0/).

Abstract— Endophytic fungi represent promising biocontrol agents due to their ecological compatibility
and production of diverse bioactive metabolites. In this study, two endophytic Nigrospora species N.
sphaerica and N. osmanthi were isolated from healthy leaves of Pluchea dioscoridis and evaluated for their
antagonistic activity against eight phytopathogenic fungi, including foliar pathogens (Alternaria sp.,
Stemphylium sp. and Myrothecium verrucaria) and soilborne pathogens (Fusarium oxysporum, F. solani,
Rhizoctonia solani, Macrophomina phaseolina, Sclerotium rolfsii). Dual culture assays revealed that N.
sphaerica exhibited superior inhibitory effects, achieving up to 60% inhibition against Alternaria sp. and
53% against M. verrucaria, alongside moderate suppression of soilborne pathogens. Correspondingly,
filtrate assays showed significant biomass reduction, particularly in foliar fungi, suggesting the presence of
potent diffusible antifungal metabolites. GC–MS profiling of Nigrospora sphaerica and N. osmanthi culture
extracts revealed the predominance of phenol, 2,4-bis (1,1-dimethylethyl)-, along with several key antifungal
metabolites. N. sphaerica, in particular, exhibited a broader chemical spectrum, producing α-linolenic acid,
arachidonic acid, lactic acid, citric acid, 2-butenedioic acid, glycerol, and aromatic hydrocarbons
compounds with well-documented antifungal properties. This highlights the value of isolating safe
endophytic fungi capable of naturally synthesizing potent bioactive metabolites for sustainable fungal
disease management. These findings highlight N. sphaerica as a strong candidate for further development in
sustainable plant disease management. However, additional in vivo and field-based investigations are
essential to validate the practical application of endophytic Nigrospora spp. as biological control agents
against fungal phytopathogens.
Keywords— Pluchea dioscoridis, Nigrospora sphaerica, biocontrol, GC–MS, antifungal activity, foliar
pathogens, endophytic fungi, culture filtrates.

I. INTRODUCTION phytoremediation of heavy metal contaminated soils,

Pluchea dioscoridis (L.) DC., commonly known as making it important for both biodiversity conservation and
“Barnûf,” is a perennial wild shrub in the family environmental restoration (Youssef and Diatta, 2023). Its
Asteraceae, naturally distributed across arid and semi-arid adaptability to harsh climates and ability to host diverse
regions of North Africa and the Middle East, including the microbial endophytes further enhance its potential as a
Nile Valley, Delta, and desert peripheries of Egypt. In model for plant microbe interaction studies under stress
addition to its extensive use in traditional medicine for conditions.
treating ailments such as colds, rheumatism, gastric ulcers, significant threat to agricultural productivity, especially in
and epilepsy, it holds notable ecological value. This species tropical and subtropical regions. Among foliar pathogens,
contributes to soil stabilization, desertification control, and Stemphylium spp., Alternaria spp., and Myrothecium

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Ahmed and Zakaria Antifungal Activity of Endophytic Nigrospora Species Isolated from Pluchea Plants against Some
Fungal Phytopathogens

verrucaria are well-known for causing leaf spots and blight II. MATERIALS AND METHODS
symptoms, which compromise photosynthetic efficiency 1. Plant Material Collection and Taxonomic Verification
and reduce overall plant growth and yield (Fravel et al.,
Field surveys were conducted during the active growing
2003). Meanwhile, soilborne fungal pathogens continue to
season of Pluchea dioscoridis (L.) DC. Both symptomatic
jeopardize root health in many economically important
plants showing typical foliar disease symptoms (necrotic
crops. Notably, species of Fusarium, such as F. oxysporum
spots, chlorotic halos, and irregular lesions) and
and F. solani, are well-established causal agents of vascular
asymptomatic, apparently healthy plants were targeted.
wilt, characterized by xylem colonization and systemic
From each site, representative samples including leaves,
blockage that ultimately leads to plant death.
stems, and roots were collected in sterile polyethylene bags
Macrophomina phaseolina is a common cause of charcoal
and transported to the laboratory under cooled conditions.
rot, particularly under heat and drought stress conditions,
Plant specimen was identified by Dr. Omran Ghaly,
while Rhizoctonia solani and Sclerotium rolfsii are
Herbarium of Desert Research Center (CAIH) with
responsible for damping-off and stem/collar rots in a wide
Identification Code: CAIH-1350-R.
host range (Fontana et al., 2021).
Although chemical fungicides have long been the mainstay 2. Isolation of Fungal Pathogens from Diseased Plants
of disease management, their excessive use health risks to and Soil
humans and non-target organisms. These challenges have Isolation procedures aimed to recover the full spectrum of
spurred the development of sustainable alternatives, with fungal pathogens associated with P. dioscoridis. Diseased
biological control emerging as a promising strategy tissues were first washed under running tap water to remove
(Fontana et al., 2021). debris, surface-sterilized in 1% sodium hypochlorite for 2
In this context, endophytic fungi microorganisms that minutes, rinsed thrice with sterile distilled water, and
colonize internal plant tissues without causing visible blotted dry on sterile filter paper. Tissue segments (5 mm²)
disease to have gained increasing attention for their ability from lesion margins were plated onto Potato Dextrose Agar
to produce diverse bioactive secondary metabolites with (PDA; Difco, USA) supplemented with streptomycin (100
antifungal properties (Manganyi, 2020). These fungi can mg/L) to suppress bacterial contamination. Plates were
suppress pathogens via competition for nutrients and space, incubated at 25 ± 2°C in darkness for 5–7 days, and
mycoparasitism, production of inhibitory metabolites, or by emerging fungal colonies were purified by hyphal-tip or
triggering systemic resistance in host plants. Among the single-spore isolation (Leslie and Summerell, 2006).
most promising endophytes, species of the genus
To broaden the pathogen panel, additional isolates were
Nigrospora have attracted attention due to their biochemical
obtained from rhizosphere soils and infected tissues of other
diversity and antagonistic potential. For example,
crops grown in proximity to P. dioscoridis, including faba
Nigrospora sp. produces phomalactone, a broad-spectrum
bean, citrus, peanut, and alfalfa. Soil samples were
antifungal compound active against several major
processed using soil dilution plating, while plant tissues
phytopathogens (Ramesha et al., 2020). More recently,
followed the same surface sterilization and plating protocol.
other metabolites such as nigrosphaeritriol and
nigrosphaerilactol have been identified from N. sphaerica, 3. Morphological Identification of Pathogens
showing potent antifungal activity and potential for Purified isolates were transferred to fresh PDA and
development as eco-friendly fungicides (Salvatore et al., incubated under controlled conditions to induce sporulation.
2024). Importantly, endophytes isolated from the same host Colony morphology, pigmentation, and growth rate were
plant are often ecologically pre-adapted, enhancing their documented, while microscopic examination of conidia,
compatibility and persistence, which supports their utility as hyphae, and reproductive structures was performed using a
long-term biological control agents (Manganyi, 2020). compound microscope (Olympus BX51). Identification to
Given these considerations, the present study aims to isolate genus level relied on morphological keys and descriptions
and characterize endophytic Nigrospora species from (Barnett and Hunter, 1998), and representative isolates were
healthy P. dioscoridis plants and evaluate their antagonistic retained for molecular identification.
activity against a spectrum of fungal phytopathogens
4. Pathogenicity Testing
isolated from diseased P. dioscoridis, soil, and other crops.
The goal is to identify ecologically compatible endophytes Pathogenicity of each isolate was confirmed using detached
with strong biocontrol potential, contributing to sustainable leaf assays (Bashyal et al., 2022). Healthy leaves of P.
crop protection while reducing dependence on synthetic dioscoridis were surface-sterilized and placed on moist
fungicides and supporting environmentally sound filter paper in sterile Petri dishes. Mycelial plugs (5 mm
agricultural practices. diameter) from actively growing cultures were placed on the
leaf surfaces; controls received sterile PDA plugs. Plates

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Ahmed and Zakaria Antifungal Activity of Endophytic Nigrospora Species Isolated from Pluchea Plants against Some
Fungal Phytopathogens

were incubated at 25 °C under a 12 h photoperiod, and filtrate was incorporated into PDA at final concentrations of
lesion development was assessed over 5–7 days. Pathogens 5%, 10%, and 15% (v/v). Pathogen mycelial plugs were
were re-isolated from symptomatic leaves and compared placed centrally, and radial growth was measured after
morphologically to the original cultures to satisfy Koch’s incubation.
postulates. 8. Extraction and GC–MS Analysis of Bioactive
5. Isolation of Endophytic Fungi from Healthy Plants Metabolites
Endophytic fungi were isolated from symptom-free tissues The ethyl acetate extracts of the culture filtrates obtained
of P. dioscoridis collected from the same sites as diseased from liquid-grown endophytic fungi were subjected to GC–
plants. Leaves and stems were surface sterilized by MS analysis. Specifically, sterile culture filtrates of
immersion in 1% sodium hypochlorite for 2 minutes, Nigrospora osmanthi and Nigrospora sphaerica grown in
followed by 70% ethanol for 30 seconds, then rinsed in potato dextrose broth (PDB) were extracted with ethyl
sterile distilled water. After blotting dry, tissue segments (5 acetate in a 1:1 (v/v) ratio under vigorous shaking for 30
mm²) were plated on PDA supplemented with streptomycin. minutes. The organic layers were separated, dried over
Plates were incubated at 25 °C for up to 10 days, with daily anhydrous sodium sulphate to remove residual moisture,
monitoring for emerging fungal colonies from internal and subsequently concentrated under reduced pressure
tissues. Colonization frequency (%) was calculated as the using a rotary evaporator to yield the crude extract
proportion of tissue segments yielding fungal growth containing secondary metabolites.
relative to the total plated segments (Pavithra et al., 2020). The extracts were analysed using gas chromatography mass
6. Molecular Identification of Endophytes and Selected spectrometry (GC–MS) to identify volatile and semi-
Pathogens volatile components. Metabolites were tentatively
Genomic DNA was extracted from pure fungal cultures identified by comparing their mass spectral fragmentation
using the cetyltrimethylammonium bromide (CTAB) patterns and retention times with reference spectra from the
method (Doyle and Doyle, 1990). The internal transcribed NIST (National Institute of Standards and Technology)
spacer (ITS) region of rDNA was amplified by PCR using Mass Spectral Library. This method is widely accepted for
primers ITS1 and ITS4. PCR reactions were performed in a metabolite profiling of fungal endophytes due to its
25 μL mixture containing 1× PCR buffer, 1.5 mM MgCl₂, sensitivity and reproducibility (Devi et al., 2010; Kaur and
0.2 mM dNTPs, 0.4 μM of each primer, 1 U Taq DNA Arora, 2009; Strobel and Daisy, 2003).
polymerase, and 50 ng template DNA. Thermal cycling Such an approach enables the characterization of complex
conditions included initial denaturation at 95 °C for 5 min, metabolite mixtures and facilitates the identification of
followed by 35 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 bioactive compounds that may be involved in antifungal or
°C for 1 min, and a final extension at 72 °C for 7 min. antimicrobial activities (Saranraj and Sivasakthi, 2014;
PCR products were purified and sequenced commercially. Narvaez-Barragan et al., 2020).
Sequences were aligned and analyzed using BLASTn 9. Statistical Analysis
against the NCBI GenBank database. Phylogenetic trees All experiments were conducted in triplicate following a
were constructed in MEGA X to confirm species-level completely randomized design. Data were analyzed by one-
identification. way ANOVA using SPSS software. Mean comparisons
7. In Vitro Antagonistic Activity Assays were performed using Tukey’s HSD test at a significant
Two complementary approaches were used to evaluate the level of p < 0.05.
antagonistic potential of endophytes against the isolated
phytopathogens: III. RESULTS AND DISCUSSION
a) Dual culture assay following Skidmore and Dickinson 1. Isolation and Identification of Pathogenic Fungi
(1976), 5 mm mycelial plugs of the pathogen and endophyte
Field surveys of Pluchea dioscoridis plants showing foliar
were placed 6 cm apart on PDA plates. Plates were
and stem lesions resulted in the isolation of two consistent
incubated at 25 °C, and radial growth was measured after 7
pathogenic fungi directly associated with symptomatic
days. Percentage inhibition was calculated relative to
tissues: Alternaria sp., producing olive-green to black
pathogen growth on control plates.
colonies with beaked conidia, and Stemphylium sp., which
b) Culture filtrate assay endophytes were grown in Potato exhibited slow-growing, dark brown to black colonies with
Dextrose Broth (PDB) at 120 rpm and 25 °C for 14 days. multicellular muriform conidia. Both isolates successfully
Culture broth was filtered through Whatman No.1 paper, reproduced characteristic leaf spot and blight symptoms on
then sterilized using a 0.22 μm membrane filter. The sterile

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Ahmed and Zakaria Antifungal Activity of Endophytic Nigrospora Species Isolated from Pluchea Plants against Some
Fungal Phytopathogens

detached leaves under controlled pathogenicity assays, vascular wilts, charcoal rot, and damping-off diseases, also
confirming their pathogenic role on P. dioscoridis. Myrothecium verrucaria as Foliar/stem blight causal agent
To extend the relevance of the antifungal screening, a in a wide range of crops.
collection of agriculturally important soilborne fungi was This pathogen panel, comprising host-specific foliar
established from rhizospheric soils or infected plant debris pathogens and ecologically relevant soilborne fungi, was
in nearby fields cultivated with crops such as alfalfa, peanut, selected to support a comprehensive assessment of potential
citrus, and faba bean. The isolated pathogens included biocontrol agents, focusing on their antagonistic capabilities
Fusarium oxysporum, F. solani, Macrophomina against diverse phytopathogens of both medicinal and
phaseolina, Rhizoctonia solani, Sclerotium rolfsii, and all of economic significance.
which are well-documented causal agents of root rots,

Fig. 1: Morphological appearance of Pluchea dioscoridis plants collected during field survey.
(A, B) Healthy plant showing normal foliage and stem development. (C–F) Naturally infected plants exhibiting foliar and
stem symptoms caused by fungal pathogens isolated in this study.

Table 1. Pathogenic fungi isolated from P. dioscoridis, their source, morphology, and growth rate on PDA
Colony Growth Rate
Source of Key Microscopic
Pathogen Morphology (mm/day) ± Disease Type
Isolation Features
on PDA SE
Olive green to Muriform, beaked Foliar (leaf
Alternaria sp. Diseased leaves 5.6 ± 0.3
black conidia spot)
Stemphylium Greyish, Branched conidiophores, Foliar (leaf
Diseased leaves 3.3 ± 0.2
sp. compact clusters of conidia spot)
Whitish with
Myrothecium Cylindrical conidia in Foliar/stem
Diseased stems green 5.8 ± 0.2
verrucaria sporodochia blight
sporodochia
Sickle-shaped
Fusarium Cottony white, macroconidia,
Diseased roots 6.9 ± 0.2 Vascular wilt
oxysporum pinkish reverse microconidia in false
heads

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Ahmed and Zakaria Antifungal Activity of Endophytic Nigrospora Species Isolated from Pluchea Plants against Some
Fungal Phytopathogens

Elliptical macroconidia,
Fusarium Soil (peanut Cream to pale Root rot, stem
abundant 6.5 ± 0.3
solani field) brown canker
chlamydospores
Macrophomina Dark grey, Numerous black
Alfalfa roots 7.0 ± 0.2 Charcoal rot
phaseolina fluffy microsclerotia
Rhizoctonia Soil (citrus Brown, web- Right-angled branching Root rot,
8.2 ± 0.3
solani orchard) like mycelium hyphae damping-off
White
Sclerotium Peanut stem mycelium, Stem base rot,
Tan to brown sclerotia 7.7 ± 0.2
rolfsii base abundant wilt
sclerotia

2. Isolation and Preliminary Identification of feature of dematiaceous fungi. No signs of pathogenicity

Endophytic Fungi were observed on host tissues at the time of sampling,
Endophytic fungal isolates were successfully recovered supporting their endophytic nature.
from surface-sterilized tissues of Pluchea dioscoridis plants To confirm their identity and differentiate between closely
that appeared asymptomatic in the field. Initial related taxa, molecular characterization was subsequently
morphological and microscopic examinations revealed that performed using ITS-rDNA sequencing. This molecular
the dominant isolates belonged to the genus Nigrospora. approach provided accurate species-level identification,
The colonies exhibited rapid growth with a characteristic confirming the isolates as Nigrospora osmanthi and
cottony to fluffy aerial mycelium, initially white turning Nigrospora sphaerica. The molecular data were further
grayish-black with age. Microscopically, the isolates were supported by phylogenetic analyses aligning the isolates
identified by their dark, globose to subglobose, single- within established Nigrospora clades.
celled conidia, typically borne laterally on short, hyaline These results underscore the importance of integrating
conidiophores (Fig. 2). The conidia measured morphological and molecular tools for the accurate
approximately 10–15 µm in diameter, consistent with identification of endophytic fungi and support the potential
taxonomic descriptions of Nigrospora spp. (Ellis, 1971; of Nigrospora spp. as candidates for biocontrol
Kirk, P.M., 1991; Barnett and Hunter, 1998,). applications.
The vegetative mycelium consisted of branched, septate,
and hyaline hyphae that later turned pigmented, a common

Fig. 2: Morphological and cultural characteristics of endophytic Nigrospora isolates from Pluchea dioscoridis; (A) Colony
morphology and microscopic features of Nigrospora osmanthi, showing septate hyphae and typical black, single-celled
conidia; (B) Colony morphology and microscopic features of Nigrospora sphaerica, displaying branched hyphae and
globose to ellipsoid conidia.

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Ahmed and Zakaria Antifungal Activity of Endophytic Nigrospora Species Isolated from Pluchea Plants against Some
Fungal Phytopathogens

3. Pathogenicity Testing lesions (9.6 ± 0.5 mm) and exhibited a slightly longer
Given the biological and ecological divergence between incubation period of seven days. Based on these
foliar and soilborne pathogens, pathogenicity assessment observations, the pathogenicity ranking among foliar
was selectively applied only to the isolates suspected of isolates was (Alternaria sp.; Myrothecium verrucaria;
causing foliar diseases in Pluchea dioscoridis. These Stemphylium sp.). This ranking aligns with known
included Alternaria sp. and Stemphylium sp., all of which pathogenic behaviour of these fungi in various host plants,
were originally recovered from symptomatic leaves and where Alternaria species are typically aggressive
stems of naturally infected P. dioscoridis plants. necrotrophs (Kusaba and Tsuge, 1995; Shoemaker and
Babcock, 1992).
3.1. Detached Leaf Assay for Foliar Pathogens:
Pathogenicity of the three foliar isolates was tested using the 3.2. Rationale for Excluding Root Pathogens from
detached leaf assay. Healthy, surface-sterilized leaves of P. Pathogenicity Assay: In contrast, the five soilborne fungi
dioscoridis were placed on moist filter paper in sterile Petri isolated from rhizospheric soil and nearby crops (Fusarium
dishes and inoculated with mycelial plugs from 7-day-old oxysporum, F. solani, Macrophomina phaseolina,
cultures of each fungal isolate. Control leaves received Rhizoctonia solani, and Sclerotium rolfsii) were not
sterile PDA plugs. Inoculated leaves were incubated at 25 ± subjected to detached-leaf assays. This was due to their
2°C under a 12-hour photoperiod for 10 days, during which fundamentally different infection biology, which involves
symptoms were monitored and lesion development root colonization and systemic vascular invasion, rendering
recorded. The results, summarized in Table 2, showed clear leaf-based assays ineffective for evaluating their virulence.
variation in virulence among the tested isolates. Alternaria Moreover, the pathogenicity of these fungi is well-
sp. caused the largest mean lesion diameter (15.1 ± 0.8 mm), established in the literature, with multiple studies
followed closely by Myrothecium verrucaria (13.6 ± 0.7 confirming their role in causing wilting, root rot, and
mm), both producing lesions within six days post- damping-off symptoms in various economically important
inoculation. Stemphylium sp.. induced significantly smaller crops (Leslie and Summerell, 2006; Kaur et al., 2012).
Table 2. Pathogenicity of foliar pathogens on detached P. dioscoridis leaves
Mean Lesion Diameter Incubation Period Pathogenicity
Pathogen
(mm) ± SE (days) Rank
Alternaria sp. 15.1 ± 0.8 6 2
Myrothecium verrucaria 13.6 ± 0.7 6 2
Stemphylium sp. 9.6 ± 0.5 7 3

4. Antagonistic Activity of Endophytic Nigrospora verrucaria. This supports earlier findings that foliar
Isolates endophytes are more competitive in the phyllosphere due to
4.1 Dual Culture Assay niche overlaps with foliar pathogens (Strobel and Daisy,
2003; Mousa and Raizada, 2013; Deshmukh et al., 2018).
To assess the antagonistic efficacy of endophytic
In contrast, root infecting fungi such as Sclerotium rolfsii.
Nigrospora isolates against phytopathogenic fungi, a dual
Macrophomina phaseolina and Rhizoctonia solani
culture assay was employed. The test included three foliar
exhibited markedly lower inhibition 32.3%, 24.2%, and
pathogens (Alternaria sp., Stemphylium sp. and
25.3% (Table 3), which could be due to their aggressive
Myrothecium verrucaria) and soilborne pathogens
colonization strategies and physical resilience, such as
(Fusarium oxysporum, F. solani, Sclerotium rolfsii,
sclerotia formation (Dean et al., 2012). The intermediate
Macrophomina phaseolina, and Rhizoctonia solani). Each
response observed in Fusarium species suggests that
isolate of N. sphaerica and N. osmanthi was evaluated for
endophyte-derived metabolites may partially limit their
its ability to suppress the radial growth of these pathogens.
growth. Although initial inhibition zones were observed
The results revealed that Nigrospora sphaerica exhibited
around the endophytic isolates, further incubation revealed
superior antagonistic activity compared to N. osmanthi
that the endophytes continued to grow, progressively
against all tested pathogens, indicating a species dependent
occupying the space and suppressing pathogen
difference in biocontrol efficacy. Notably, foliar pathogens
development through competitive exclusion. This suggests
showed higher susceptibility to both endophytes,
that inhibition was not only due to static antagonism but also
particularly to N. sphaerica, which achieved inhibition rates
involved active overgrowth and space competition (Fig. 3).
exceeding 60% in Alternaria sp. and 53.9% in Myrothecium

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Ahmed and Zakaria Antifungal Activity of Endophytic Nigrospora Species Isolated from Pluchea Plants against Some
Fungal Phytopathogens

These findings underscore the ecological relevance of for foliar disease management in Pluchea dioscoridis and
endophyte-host interactions and support the use of N. possibly other hosts.
sphaerica as a potential biocontrol candidate, particularly
Table 3. Radial Growth Inhibition of Pathogenic Fungi by Nigrospora spp. in Dual Culture Assays
Growth
Growth with Growth with
Pathogen Alone Inhibition % Inhibition %
N. osmanthi N. sphaerica
(mm/day)
Alternaria sp. 9.6 ± 0.3 6.3 ± 0.2 34.4% 4.2 ± 0.3 56.3%
Myrothecium verrucaria 8.9 ± 0.4 5.8 ± 0.2 34.8% 4.1 ± 0.3 53.9%
Stemphylium sp. 7.8 ± 0.4 5.9 ± 0.2 24.4% 3.8 ± 0.3 51.3%
Fusarium oxysporum 10.1 ± 0.4 7.6 ± 0.3 24.8% 5.7 ± 0.4 43.6%
Fusarium solani 9.8 ± 0.3 7.3 ± 0.2 25.5% 5.6 ± 0.3 42.9%
Sclerotium rolfsii 9.3 ± 0.3 7.7 ± 0.2 17.2% 6.3 ± 0.4 32.3%
Macrophomina
9.5 ± 0.4 8.0 ± 0.3 15.8% 7.2 ± 0.2 24.2%
phaseolina
Rhizoctonia solani 8.7 ± 0.3 7.4 ± 0.3 14.9% 6.5 ± 0.3 25.3%

Fig. 3: Dual culture assay showing antifungal activity of endophytic Nigrospora isolates against phytopathogenic fungi.
(A) Nigrospora osmanthi; (B) Nigrospora sphaerica. Although initial inhibition zones were observed around the
endophytic isolates, further incubation revealed that the endophytes continued to grow, progressively occupying the
space and suppressing pathogen development through competitive exclusion. This suggests that inhibition was not only
due to static antagonism but also involved active overgrowth and space competition.

4.2 Effect of Culture Filtrates of Nigrospora spp. on growth rates and percentage inhibition values for each
Growth of Pathogens pathogenic fungus in response to the culture filtrates, along
To further assess the antagonistic efficacy of Nigrospora with Duncan’s multiple range test letters indicating
osmanthi and Nigrospora sphaerica, culture filtrate assays statistically significant differences (P ≤ 0.05).
were conducted to evaluate the extracellular inhibitory The results presented in Table 4 show clear differences in
potential of these endophytes against a range of the antifungal activity of culture filtrates between N.
phytopathogens. Filtrates obtained from liquid cultures of sphaerica and N. osmanthi. In all tested pathogens, filtrates
each isolate were incorporated into PDA medium at from N. sphaerica demonstrated significantly stronger
standardized concentrations. The radial growth of each inhibitory effects compared to N. osmanthi, suggesting a
pathogen was then recorded and compared to controls after superior production of bioactive metabolites by this isolate.
7 days of incubation. Table 4 summarizes the mean radial This intra-genus variability in antagonistic capacity is

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Ahmed and Zakaria Antifungal Activity of Endophytic Nigrospora Species Isolated from Pluchea Plants against Some
Fungal Phytopathogens

consistent with earlier studies, which highlighted that antifungal compounds (Dean et al., 2012; Khaledi and
secondary metabolite production among endophytes can Taheri, 2016).
vary markedly depending on strain and environmental Intermediate suppression was observed for Fusarium
adaptation (Kharwar et al., 2011; Strobel, 2003). species, which are vascular pathogens with systemic
Foliar pathogens such as Alternaria sp., Myrothecium colonization behavior. The moderate inhibition (~30–35%)
verrucaria, and Stemphylium sp. were among the most suggests that while the endophytes metabolites interfere
susceptible to culture filtrates, with inhibition rates with hyphal development, they may not fully suppress more
exceeding 40% in the case of N. sphaerica. This is likely aggressive soilborne pathogens. Such partial inhibition
due to the shared ecological niche between these pathogens aligns with previous findings in which Fusarium spp.
and the endophytes, which may facilitate competitive showed variable responses to fungal filtrates, depending on
exclusion and higher local accumulation of antifungal the bioactive profile of the antagonistic strain (Palaniyandi
compounds (Schulz and Boyle, 2005; Ghorbanpour et al., et al., 2013). Overall, the filtrate assay confirms the
2018). antagonistic potential of Nigrospora endophytes,
In contrast, soilborne pathogens such as Macrophomina particularly N. sphaerica, against a range of
phaseolina and Rhizoctonia solani showed the lowest levels phytopathogens. These findings reinforce their biocontrol
of growth inhibition, not exceeding 16% even under N. capacity and ecological compatibility with the aerial parts
sphaerica filtrate treatment. These fungi are known for their of Pluchea dioscoridis, making them promising candidates
robust sclerotial structures and tolerance to environmental for further development as biological control agents.
stress, which may reduce the efficacy of diffusible
Table 4. Effect of Culture Filtrates of Nigrospora spp. on Radial Growth of Pathogenic Fungi (mm/day) and Growth
Inhibition (%).
Control N. osmanthi N. sphaerica
Filtrate<br> Filtrate<br> Inhibition
Pathogenic Fungi Growth<br> Inhibition (%)
(%)
(mm/day) (mm/day) (mm/day)
Alternaria sp. 8.0 a 5.2 c 35.0 4.4 d 45.0
Myrothecium verrucaria 7.6 a 5.3 c 30.3 4.5 d 40.8
Stemphylium sp. 7.4 a 5.5 c 25.7 4.8 d 35.1
Fusarium solani 8.2 a 6.1 bc 25.6 5.3 c 35.4
Fusarium oxysporum 8.1 a 6.3 bc 22.2 5.6 c 30.9
Sclerotium rolfsii 7.8 a 6.6 b 15.4 6.2 bc 20.5
Rhizoctonia solani 8.0 a 7.0 ab 12.5 6.7 b 16.3
Macrophomina
7.9 a 7.1 ab 10.1 6.8 b 13.9
phaseolina

Effect of Fungal Culture Filtrates of Nigrospora spp. on observed against foliar pathogens such as Alternaria sp. and
Dry Biomass of Phytopathogenic Fungi Myrothecium verrucaria, with their dry weights dropping to
To explore the metabolite-based antagonistic potential of approximately 0.29 g/plate and 0.26 g/plate, respectively.
the endophytic Nigrospora isolates (N. osmanthi and N. This supports the hypothesis that foliar-derived endophytes
sphaerica), culture filtrate assays were conducted, where may biosynthesize metabolites specifically adapted to target
the dry biomass of various phytopathogenic fungi served as aerial pathogens, consistent with findings by Amin et al.
a quantitative measure of growth inhibition. This approach (2021) and Gakuubi et al. (2022).
distinguishes the chemical inhibitory effects mediated by Conversely, soilborne pathogens like Rhizoctonia solani
secondary metabolites from direct mycelial competition. and Macrophomina phaseolina showed more moderate
As illustrated in Fig. 4, both isolates significantly reduced reductions (dry weights 0.51–0.55 g/plate), which could be
fungal biomass compared to the control. N. sphaerica attributed to their well-documented resilience and
consistently exhibited a greater inhibitory effect across all enzymatic detoxification systems (Khaledi et al., 2016;
tested pathogens, corroborating its superior performance in Singh and Yadav, 2020).
dual culture assays. The most pronounced suppression was

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Ahmed and Zakaria Antifungal Activity of Endophytic Nigrospora Species Isolated from Pluchea Plants against Some
Fungal Phytopathogens

Intermediate responses were recorded for Fusarium These results suggest that while N. sphaerica exhibits
oxysporum and F. solani (0.41–0.45 g/plate), indicating a broader antifungal metabolite diversity and potency, its
partial susceptibility likely involving disruption of cellular effectiveness is more apparent against foliar pathogens. The
processes such as membrane integrity or secondary reduced biomass levels confirm that the secreted bioactive
metabolism (Larran et al., 2016). compounds contribute substantially to fungal inhibition and
reinforce the potential use of these endophytes in integrated
disease management strategies.

Fig. 4: Effect of Culture Filtrates of Nigrospora spp. on Dry Biomass of Phytopathogenic Fungi (g/plate)

4.3 Molecular Identification of Endophytic Nigrospora Trichosphaeriales, and genus Nigrospora. These results
Isolates affirm the identification of H1 as Nigrospora osmanthi.
To confirm the taxonomic identity of the two endophytic The second isolate, designated H2, yielded a 575 bp ITS
fungal isolates obtained from healthy Pluchea dioscoridis sequence, which showed 99% identity (549/552 bp) with
leaves, molecular identification was carried out using Nigrospora sphaerica (GenBank accession number
internal transcribed spacer (ITS) rDNA sequencing. MG669225.1), also with 100% coverage. The isolate was
Genomic DNA was extracted from actively growing taxonomically assigned to Nigrospora sphaerica based on
mycelia, and amplification of the ITS region was performed sequence similarity, supporting its classification within the
using the universal primers (ITS1: 5'-TCC GTA GGT GAA same taxonomic lineage as H1.
CCT GCG G-3' ; ITS4: 5'-TCC TCC GCT TAT TGA TAT Interpretation and Relevance, the high sequence identity
GC-3'). The amplified PCR products were purified and and coverage of both isolates to their respective type strains
sequenced bidirectionally. The resulting sequences were support their accurate species-level identification. These
analyzed using the BLASTn algorithm against the NCBI findings align with previous studies demonstrating the
GenBank database to determine species-level similarity utility of ITS sequencing for delimiting Nigrospora species
(Fig. 5). (Wang et al., 2018; Gomes et al., 2013). Importantly, both
The first isolate, designated H1, produced a high-quality isolates were recovered from asymptomatic P. dioscoridis
sequence of 592 base pairs. BLASTn analysis revealed a tissues and did not produce symptoms under pathogenicity
99% identity (567/571 bp) with Nigrospora osmanthi assays, confirming their endophytic. The confirmed identity
(GenBank accession number MH645207.1). The query of these isolates allows for reliable attribution of the
coverage was 100%, confirming a high-confidence match. observed biocontrol effects in subsequent antagonism and
Phylogenetic placement aligned H1 within the filtrate assays. Moreover, it supports future work exploring
Trichosphaeriaceae family under the order metabolite profiling or gene expression patterns related to
antifungal activity (Zhao et al., 2011).

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Ahmed and Zakaria Antifungal Activity of Endophytic Nigrospora Species Isolated from Pluchea Plants against Some
Fungal Phytopathogens

Fig. 5: Phylogenetic dendrograms illustrating the molecular identification of two endophytic fungal isolates from Pluchea
dioscoridis based on ITS region sequencing. (A): Isolate H1 clustered with Nigrospora osmanthi (GenBank accession no.
MH645207.1) with 99% sequence identity. (B): Isolate H2 clustered with Nigrospora sphaerica (GenBank accession no.
MG669225.1) with 99% sequence identity. The trees were constructed using standard BLASTn alignment and similarity
clustering NCBI database sequences. Bootstrap values indicate the confidence of clustering for each node.

4.4 GC–MS-Based Metabolomic Profiling of chemical diversity and the low representation of
Endophytic Nigrospora spp. Culture Filtrates complementary bioactive compounds likely contribute to
Nigrospora species, GC–MS analysis was performed on the isolate’s moderate antifungal efficacy. Phenol
ethyl acetate extracts of culture filtrates from Nigrospora derivatives such as 2,4-di-tert-butylphenol have been shown
osmanthi and N. sphaerica. The analysis enabled to inhibit fungal spore germination and growth by
identification of several volatile and semi-volatile disrupting membrane (Devi et al., 2010), but their efficacy
compounds, many with reported antimicrobial functions, may depend on synergistic interactions with other
providing a chemical basis for the observed variation in metabolites, which appear minimal in this strain.
biocontrol activity. 4.4.2 Metabolite Profile of Nigrospora sphaerica
4.4.1 Metabolite Profile of Nigrospora osmanthi In contrast, GC–MS profiling of N. sphaerica revealed a
The GC–MS chromatogram of N. osmanthi revealed 24 total of 15 distinct peaks, reflecting a notably richer
peaks with retention times ranging from 8.447 to 37.687 chemical diversity and more balanced distribution of
minutes. The chemical composition was dominated by a metabolite intensities. The predominant compound detected
single compound at Rt = 34.722 min (48.48%), identified as was phenol, 2,4-bis(1,1-dimethylethyl)- (Rt = 34.773 min,
phenol, 2,4-bis(1,1-dimethylethyl)-, a compound known for 100%), followed by benzene, 1,3-bis(1,1-dimethylethyl)-
its broad-spectrum antimicrobial and antioxidant properties (Rt = 35.789 min, 49.8%), both of which are lipophilic
(Kaur and Arora, 2009). Other minor compounds included aromatic compounds known for their membrane-disruptive
1-monopalmitin (Rt = 29.397 min, 10.09%) and citric acid properties. Beyond these major constituents, the isolate also
(Rt = 22.874 min, 35.89%), (Fig. 6). produced a range of bioactive metabolites including lactic
acid, which is recognized for its acidifying effect that
Although a relatively high number of peaks was detected,
creates unfavourable conditions for pathogen survival, and
the predominance of a single compound indicates a
glycerol, a metabolite often associated with cellular stress
metabolically biased output concentrated along a narrow
responses and osmotic balance. Organic acids such as citric
biosynthetic pathway. While this major phenolic metabolite
acid and 2-butenedioic acid, along with β-D-(+)-
may possess antifungal potential, the overall lack of

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Ahmed and Zakaria Antifungal Activity of Endophytic Nigrospora Species Isolated from Pluchea Plants against Some
Fungal Phytopathogens

xylopyranose, were also present and may enhance its strong potential for incorporation into integrated pest
antifungal activity through mechanisms involving metal ion management (IPM) strategies.
chelation, pH reduction, or interference with pathogen Bioactive Metabolites and Antifungal Potential: The
metabolism. Additionally, the detection of α-linolenic acid GC–MS profiles of both Nigrospora osmanthi and N.
and arachidonic acid fatty acids known to inhibit biofilm sphaerica revealed the presence of several volatile and
formation and compromise fungal membranes further semi-volatile metabolites with reported antifungal
supports the potential of N. sphaerica as a multifaceted properties. Most notably, 2,4-di-tert-butylphenol (also
source of antifungal compounds. known as Phenol, 2,4-bis(1,1-dimethylethyl)-) was
The presence of multiple moderately to highly abundant identified as the major compound in both extracts, with a
compounds, spanning both polar and non-polar classes, relative abundance exceeding 98% in N. osmanthi and
suggests that N. sphaerica harbors a broad-spectrum 100% in N. sphaerica. This phenolic compound has been
metabolic. These compounds likely act synergistically, widely documented for its antimicrobial and antifungal
targeting various aspects of fungal physiology such as properties, attributed to its membrane-disrupting effects and
membrane permeability, enzymatic activit. This chemical interference with ergosterol synthesis in fungal cells (Devi
richness is consistent with the enhanced antifungal activity et al., 2010; Huang et al. 2007; Kaur and Arora, 2009;
observed for N. sphaerica in both dual culture and filtrate Ghorbanpour, et al., 2018).
assays. Additional metabolites detected exclusively or at higher
Comparative Interpretation: Although N. osmanthi intensity in N. sphaerica include Benzene, 1,3-bis(1,1-
exhibited a greater number of chromatographic peaks, its dimethylethyl)-, which has been associated with antifungal
metabolite distribution was heavily dominated by a single activity via disruption of lipid bilayers (Saranraj and
phenolic compound, which accounted for over 98% of the Sivasakthi, 2014), and lactic acid, a known inhibitory agent
total peak area. In contrast, the chemical profile of N. that lowers extracellular pH and impairs fungal respiration
sphaerica was more balanced and functionally diverse, with (Magnusson and Schnürer, 2001). The detection of citric
several bioactive metabolites contributing substantially to acid and glycerol in both isolates may also contribute to
the overall composition of the extract. This functional antifungal efficacy through osmotic stress induction and
chemical diversity likely underpins the superior biocontrol chelation of essential metal ions (Jung et al., 2003;
efficacy of N. sphaerica, as confirmed in bioassays. The Hallsworth and Magan, 1995).
results highlight that not only the number of metabolites, Furthermore, N. sphaerica produced fatty acid derivatives
but their relative abundance and functional such as α-linolenic acid and arachidonic acid, both of which
complementarity are critical in determining antifungal are recognized for their fungitoxicity and ability to disrupt
potential. membrane integrity in phytopathogens (Walters et al.,
The observed variation in metabolite expression between 2004; Narvaez-Barragan et al., 2020). These metabolites,
the two fungal strains may stem from inherent genetic especially when present in combination, may account for
differences in their secondary metabolic pathways, as well the enhanced and broader-spectrum antifungal activity
as disparities in host-endophyte interactions during the exhibited by N. sphaerica in bioassays. The presence and
initial isolation process. Additionally, environmental and distribution of these metabolites not only explain the
culture conditions are known to influence metabolic flux superior biocontrol potential of N. sphaerica but also
and can significantly shape the profile of secondary highlight the utility of metabolomic profiling in guiding the
metabolites produced (Strobel, 2003; Kusari et al., 2012). selection of promising endophytes for antifungal
These findings underscore the importance of integrating applications. Future work involving compound purification
metabolomic profiling with antifungal bioassays to guide and structure activity relationship (SAR) studies is
the selection of promising fungal isolates for further warranted to confirm the specific roles of these bioactives
development in biological control programs. The detection in pathogen inhibition.
of known antifungal compounds in N. sphaerica highlights

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Ahmed and Zakaria Antifungal Activity of Endophytic Nigrospora Species Isolated from Pluchea Plants against Some
Fungal Phytopathogens

Fig. 6: GC–MS chromatograms of ethyl acetate extracts from culture filtrates of endophytic Nigrospora osmanthi showing
24 major peaks

Table 5. GC–MS Detected Peaks in Culture Filtrate of N. osmanthi

Area
Peak RT Name Formula Area
Sum %
1 8.447 Lactic Acid, 2TMS derivative C₉H₂₂O₃Si₂ 324397.63 2.77
2 14.951 Glycerol, 3TMS derivative C₁₂H₃₂O₃Si₃ 257515.76 2.2
3 18.783 Propylene glycol, 2TMS derivative C₉H₂₄O₂Si₂ 133172.22 1.14
4 19.041 Arachidonic acid, TMS derivative C₂₃H₄₀O₂Si 58312.6 0.5
5 19.155 L-5-Oxoproline,2TMS derivative C₁₁H₂₃NO₃Si₂ 1330542.5 11.36
6 19.997 Succinic acid (tms) C₁₀H₂₂O₄Si₂ 51776.68 0.44
7 21.014 2,4-Hexadien-1-ol C₆H₁₀O 29310.13 0.25
8 21.895 Xylitol, 5TMS derivative C₂₀H₅₂O₅Si₅ 637209.12 5.44
9 22.464 Glyceric acid, 3TMS derivative C₁₂H₃₀O₄Si₃ 104564.7 0.89
10 22.684 .beta.-Arabinopyranose, 4MS derivative C₁₇H₄₂O₅Si₄ 524338.61 4.48
11 22.874 Citric acid, 4TMS derivative C₁₈H₄₀O₇Si₄ 477565.38 4.08
12 23.147 Methyl .alpha.-D-glucofuranoside, 4TMS derivative C₁₉H₄₆O₆Si₄ 1306674.3 11.16
13 23.42 2-Butenedioic acid, (Z)-, 2TMS derivative C₁₀H₂₀O₄Si₂ 828003.87 7.07
14 23.663 D-Galactose, 5TMS derivative C₂₁H₅₂O₆Si₅ 942605.46 8.05
15 23.761 .beta.-D-(+)-Xylopyranose, 4TMS derivative C₁₇H₄₂O₅Si₄ 1090960.2 9.32
16 24.057 Ribitol, 5TMS derivative C₂₀H₅₂O₅Si₅ 1121786.3 9.58
17 24.611 β-D-glucose 5-TMS derivative C₂₁H₅₂O₆Si₅ 825202.82 7.05
18 24.93 α-Linolenic acid, TMS derivative C₂₁H₃₈O₂Si 109200.24 0.93
19 29.438 1-Monopalmitin, 2TMS derivative C₂₅H₅₄O₄Si₂ 134196.3 1.15
20 33.521 Phenol, 2,6-bis(1,1-dimethylethyl)- C₁₄H₂₂O 324724.18 2.77
21 34.773 Phenol, 2,4-bis(1,1-dimethylethyl)-, phosphite (3:1) C₄₂H₆₃O₃P 645042.4 5.51
22 35.782 Benzene, 1,3-bis(1,1-dimethylethyl)- C₁₄H₂₂ 395442.75 3.38
23 37.231 Silicic acid, diethyl bis(trimethylsilyl) ester C₁₀H₂₈O₄Si₃ 26890.61 0.23
24 37.785 1,4-Benzenediol, 2,6-bis(1,1-dimethylethyl)- C₁₄H₂₂O₂ 28083.68 0.24

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Ahmed and Zakaria Antifungal Activity of Endophytic Nigrospora Species Isolated from Pluchea Plants against Some
Fungal Phytopathogens

Fig. 7: GC–MS chromatograms of ethyl acetate extracts from culture filtrates of endophytic Nigrospora sphaerica
displaying 15 major peaks

Table 6. GC–MS Detected Peaks in Culture Filtrate of N. sphaerica

Area
Peak RT Name Formula Area
Sum %
1 8.66 Lactic Acid, 2TMS derivative C₉H₂₂O₃Si₂ 205574.63 7.94
2 14.966 Glycerol, 3TMS derivative C₁₂H₃₂O₃Si₃ 192181.08 7.43
3 19.041 Arachidonic acid, TMS derivative C₂₃H₄₀O₂Si 36773.52 1.42
4 21.371 2,4-Hexadien-1-ol C₆H₁₀O 25933.24 1
5 22.873 Citric acid, 4TMS derivative C₁₈H₄₀O₇Si₄ 110045.65 4.25
6 23.116 Methyl α-D-glucofuranoside, 4TMS derivative C₁₉H₄₆O₆Si₄ 22363.69 0.86
7 23.412 2-Butenedioic acid, (Z)-, 2TMS derivative C₁₀H₂₀O₄Si₂ 146200.52 5.65
8 23.685 D-Galactose, 5TMS derivative C₂₁H₅₂O₆Si₅ 68840.04 2.66
9 23.792 β-D-Xylopyranose, 4TMS derivative C₁₇H₄₂O₅Si₄ 149211.9 5.77
10 24.019 Ribitol, 5TMS derivative C₂₀H₅₂O₅Si₅ 20109.93 0.78
11 24.9 α-Linolenic acid, TMS derivative C₂₁H₃₈O₂Si 184475.29 7.13
12 29.438 1-Monopalmitin, 2TMS derivative C₂₅H₅₄O₄Si₂ 203564.46 7.87
13 34.773 Phenol, 2,4-bis(1,1-dimethylethyl)-, phosphite (3:1) C₄₂H₆₃O₃P 798110.29 30.84
14 35.789 Benzene, 1,3-bis(1,1-dimethylethyl)- C₁₄H₂₂ 397456.94 15.36
15 36.23 Cyclobarbital C₁₂H₁₆N₂O₃ 26928.39 1.04

IV. CONCLUSION exhibited stronger antifungal activity, particularly against

This study provides compelling evidence for the foliar pathogens such as Alternaria sp. (inhibition rate
antagonistic potential of endophytic Nigrospora species >60%) and Myrothecium verrucaria (54%), whereas N.
isolated from Pluchea dioscoridis against a spectrum of osmanthi showed moderate inhibition across all tested
foliar and soilborne phytopathogens. Two isolates fungi.
Nigrospora osmanthi and N. sphaerica were recovered endophytic fungi. N. sphaerica exhibited a chemically
from symptomatic leaf tissues and molecularly confirmed diverse and functionally rich profile, encompassing a broad
via ITS-rDNA sequencing. In vitro dual culture and culture spectrum of volatile and semi-volatile metabolites. These
filtrate bioassays revealed that N. sphaerica consistently included phenolic compounds such as 2,4-di-tert-

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Ahmed and Zakaria Antifungal Activity of Endophytic Nigrospora Species Isolated from Pluchea Plants against Some
Fungal Phytopathogens

butylphenol, organic acids like lactic and citric acids, sugar agriculture. Applied Microbiology and Biotechnology, 66(4),
alcohols, and polyunsaturated fatty acids including α- pp.439–445.
linolenic and arachidonic acids all of which are known for [6] Dean, R., Van Kan, J.A.L., Pretorius, Z.A., Hammond-
Kosack, K.E., Di Pietro, A., Spanu, P.D., Rudd, J.J.,
their pronounced antifungal and antimicrobial activities. In
Dickman, M., Kahmann, R., Ellis, J. and Foster, G.D., 2012.
contrast, the chemical profile of N. osmanthi was markedly
The top 10 fungal pathogens in molecular plant pathology.
unbalanced, being dominated by a single phenolic Molecular Plant Pathology, 13(4), pp.414–430.
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compounds. The dominance of a single metabolite in profile 2018. Endophytic fungi: a source of potential antifungal
of N. osmanthi likely contributes to its reduced spectrum of compounds. Journal of Fungi, 4(3), p.77.
antifungal activity relative to N. sphaerica. [8] Devi, P.S., Prabhakaran, N. and Arumugam, P., 2010.
Antifungal activity of 2,4-di-tert-butylphenol isolated from
The antifungal efficacy of N. sphaerica is likely attributed
Bacillus spp. Journal of Biopesticides, 3(1), pp.350–354.
to the synergistic action of its chemically diverse [9] Doyle, J.J. and Doyle, J.L., 1990. Isolation of plant DNA
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ACKNOWLEDGMENT
intracellular glycerol and erythritol enhances germination of
The authors would like to express their sincere gratitude to conidia at low water availability. Microbiology, 141(1),
Dr. Omran N. Ghaly, plant taxonomist and Director of the pp.1109–1115.
Herbarium of Desert Research Center (DRC), for his [17] Huang, W.Y., Cai, Y.Z., Xing, J., et al. (2007). A Potential
valuable contribution in the accurate identification of the Antioxidant Resource: Endophytic Fungi from Medicinal
plant specimens. The identification was carried out Plants. Economic Botany, 61, 14-30..
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following the standard taxonomic verification protocols
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