1Plant Pathology
Tamil Nadu Agricultural University Coimbatore, India
2Agricultural
Entomology, Tamil Nadu Agricultural University, Coimbatore, India
3Agronomy,
Tamil Nadu Agricultural University, Coimbatore, India
*For correspondence: sudhaa1981@gmail.com
Abstract
Biocontrol agents are the potential microbes and used
for the control of aerial and soil-borne pathogens present in all crops. An
attempt was made on identification, morphological and molecular characterization of Lasiodiplodia theobromae, a pathogen causing disease in nuts and
leaves of coconut. A virulent isolate Lasiodiplodia theobromae L26 was selected based on the growth parameters
and pycnidiospore production. Three efficient
biocontrol agents (BCAs) namely Trichoderma
asperellum, Bacillus subtilis and Streptomyces rochei, were selected for in vitro studies. Among these, T. asperellum
showed a significantly higher percentage of inhibition (81%) in dual culture
assay against L26. The inhibition was also confirmed in light microscopic
observation, the mycelium of L26 was distorted, lysis of cell wall during the
interaction. Volatile organic compounds (VOCs) emitted from BCAs inhibited the
fungal growth of L26 by 59.61–47.03% in sealed plate method. Solid-phase
microextraction GC-MS analysis revealed numerous new VOCs compounds emitted
from the BCAs, whereas the dominant compound was identified as peptaibols, 2,4-di-tert-butylphenol,
2-piperidinone.
The strength of peaks of these compounds augmented during the
interaction of BCAs with L26, the peak intensity for terpenoids was the predominant
class, followed by phenols and heterocyclic organic compound. Crude metabolite
(75 μL) of each antagonist tested through
agar well method against L26 and showed a complete inhibition. This
study demonstrated the ability of BCAs to produce volatile and nonvolatile
antifungal compounds, showing that there could a major mechanism involved in
and that will be responsible for the successful inhibition of L26 under in vitro. In future combination of these
three strains as commercial formulation may be a better management practices
for leaf blight and malformation of nuts in coconut. © 2021 Friends Science
Publishers
Keywords:
Antifungal compounds; Biocontrol agents; GC-MS; Lasiodiplodia theobromae
Introduction
Philippines is the second largest country in production,
which account for 32.67% of global production of coconut (Naik 2017). In India,
coconut plays an important role in GDP contribution of about 15,000 crore
rupees and it accounts 72% of world production. Tamil Nadu is at the top of the
list in the productivity of coconut among the states in India, (CDB-Statistics-area 2018). Coconut, a versatile crop is being
used for various uses, but in India, almost 70% of the coconut is used for the
edible purpose. There are several biotic factors which are responsible
for the drastic reduction in coconut production and productivity. The major
devastating diseases occurring on coconut in Tamil Nadu are bud rot (Phytophthora palmivora), Tanjore wilt or basal stem end rot (Ganoderma lucidum), Kerala wilt (Candidatus phytoplasma), grey blight (Pestalotiopsis palmarum), leaf blight (Lasiodiplodia theobromae)
and stem bleeding disease (Thievolopsis paradoxa). Among these, L. theobromae has become severe problem
in major coconut growing districts of Tamil Nadu namely, Coimbatore, Erode, Dindigul, Tirunelveli and Kanyakumari which, causes yield
losses of 10–25% (Johnson et al.
2014). The fungus has been act as a
secondary infector as the primary factor was due to eriophyid mite (A. guerreronis)
and this interaction was studied by Lakshmanan and Jagadeesan
(2004). Management of this malady is of immense importance by exploiting the
recently reported facts, suitable effective management strategies could be
formulated in future from the antagonists. Several biological agents viz., Trichoderma spp., (Ali et al.
2020), Aspergillus spp. Penicillium spp. (Khan and Javaid 2021a,
b) Bacillus subtilis, Pseudomonas fluorescens and Streptomyces spp. (Sharf et al. 2021) inhibit phytopathogens
growth. It is well known that, Trichoderma
spp., are successful biocontrol agents, which produced primary metabolites with
antifungal properties to induce resistance (Mukherjee et al. 2012; Khan and Javaid 2020). Macrophomina phaseolina was inhibited by the
secondary metabolites of T. viride
and it was proved by Khan et al.
(2021). The plant growth-promoting microbe, Bacillus
spp. colonizing the rhizosphere and rhizoplane region, which in turn improves vigour, plant growth and reduce disease incidence. El-Tarabily et al. (2009) and
Monteiro et al. (2017) reported that,
actinomycetes played an important role in growth promotion when compared to all
rhizosphere microbes (Ilic et al.
2007). VOCs produced by T. virens
control R. solani
under in vitro, are due to the
presence of the antifungal compound viz.,
α-cadinane, docosane
and oleic acids were detected in T.
virens. The n-butanol extract from crude culture of Bacillus spp., showed effective inhibitory activity against T. harzianum which is a pathogen observed from mushroom (Fernandes
et al. 2019). However, investigations on biopotential of
antagonists and the combined interaction with L. theobromae are still lacking. So far, not much
work has been done on identification
of volatile compounds from the BCAs with the pathogens were to be investigated.
In recent investigations the potential BCAs were identified and evaluated by
the researchers in the department of plant pathology, TNAU, Coimbatore against
few pathogens. By using the BCAs, the potent antagonist will be identified
under in vitro against L. theobromae.
This present study aims to identify the symptomatology, molecular
and morphological variations of L. theobromae in Tamil Nadu. We
employed potential antagonist for screening against the pathogen. FT-IR spectra
and Tensor 27 Infrared spectrometer was used for the compound identification
and to ascertain the biocontrol effects of volatile blends in antagonists in vitro studies were performed.
Materials and Methods
Isolation and Selection of virulent isolate
A
survey was conducted in thirty districts of Tamil Nadu to find out the
occurrence of both mite and fungal infested nuts to assess the percent
incidence, to identify the virulent isolate. Diseased nuts collected from 30
districts of Tamil Nadu during survey were used for the isolation of L. theobromae.
Isolation and purification of fungal pathogen was carried by hyphal tip
transfer procedure (Rangaswami et al. 1975) and incubated at 25 ± 3şC for 7 days (Phipps and
Porter 1998). By measuring the culture radial growth, number of pycnidia and pycnidiospore production, virulent isolate of L. theobromae, was
selected. Molecular identification of L. theobromae was carried out by the (CTAB) method described by Ma et
al. (2001) and similarity matrix was developed using the Jaccard’s
coefficient of similarity with the data matrix (Jaccard 1998).
Preparation of media and in vitro
antagonistic activity against plant pathogens
The medium was obtained from Himedia Laboratories (India) was used for culturing fungi
(Potato agar medium), nutrient agar medium was used for culturing B. subtilis and starch casein agar was used for
culturing S. rochei. In addition,
the medium (starch casein agar) was added with 25 µg mL-1
nystatin to minimize the fungal contamination. After a week the colonies
from antagonistic fungi, bacteria and S. rochei were maintained. Antifungal activity screening
was studied using potato dextrose agar medium.
Effect on radial mycelial growth by dual culture technique
The efficacy of antagonistic
organisms against the selected L. theobromae was tested by dual culture technique (Dennis
and Webster 1971). The nine biocontrol strains were obtained from the Culture
Collection Centre, Department of Plant Pathology, Tamil Nadu Agricultural
University, Coimbatore, Tamil Nadu, India to carry out the study. A nine mm
actively growing PDA culture disc of virulent isolate L. theobromae was placed onto sterilized
PDA medium, previously poured into a sterilized Petridish
approximately at a distance of 1.5 cm away from the periphery of the plate. Similarly,
nine mm culture disc of the respective fungal antagonists viz., T. asperellum
(Tv1), T. harzianum,
T. asperilloides, T. koningeopsis,
bacteria viz., Bacillus subtilis (Bs1), B. amyloliquefaciens, B.
megatherium, B. lichiniformis
and actinobacteria viz., S. rochei
were placed onto the medium at the opposite side of the culture plate separately
(Dennis and
Webster 1971). A plate with pathogen alone on the periphery served as
control. Three replications were performed to study the direct efficacy of the
antagonist. These plates were inverted and maintained at 28 ± 2°C, for five
days. Percent inhibition was calculated using the formula:
PI=C-T/C×100
Where PI = percent inhibition; C
= radial growth of the pathogen in the control plate; T = radial growth of the
pathogen in treatment.
During the study of
interaction of antagonists with L. theobromae culture was placed on slide and stain by lectophenol cotton blue, observed by Digital light
microscope at 400X to study the mode of action.
Inverted bioassay method was
carried out to know the efficacy of antifungal volatiles described by Garbeva et al.
(2014). In this method both the test pathogens and antagonists were inoculated
in same plates. To expose the pathogen to the volatile secreted by the
antagonists, the test pathogen inoculated plate was placed over the three
antagonists, and sealed; these setups were incubated at 28 ± 2°C. The growth of
hyphae was marked at regular interval and inhibition of pathogen growth was
calculated in percentage by comparing with control plate.
Inhibition of fungal growth= 100
X (1-(Ge-Ga))
Ge-mycelial growth of
pathogen in the presence of the antagonists
Ga- mycelial growth
of pathogen in the absence of the antagonists.
Preparation of inoculum and fermentation
The isolated and antifungal
tested strains of pathogen, BCAs were taken for this study for inoculums
production and fermentation. Yeast molasses broth for T. asperellum, King’B
broth for B. subtilis, Starch casein
agar medium for S. rochei
and Potato dextrose broth for L. theobromae
were used for
growing the strain for 10 days in 28°C.The well grown spore suspension was
prepared in distilled water for about 10 days. These inoculated broths of
bacteria alone were kept in a shaker at 120 rpm at 28°C for 3–5 days as stocks.
Extraction and FT-IR and GC-MS analysis of the antifungal metabolite
Antifungal metabolites from 7 days old cultures of BCAs
were extracted from cell pellets with methanol and also from culture filtrates
in ethyl acetate. The extract was dried at 40°C using a rotary evaporator and
it was suspended in 5 mL of 1% HPLC grade methanol (Intana
et al. 2005; Vinale
et al. 2006). FT-IR spectra were
recorded on Bruker, Tensor 27 Infrared spectrometer. GC-MS analysis was
performed by using FTIR and GC-MS analysis is used for the analysis of volatile
compounds in crude ethyl acetate extract of axenic and co-culture of BCAs along with L. theobromae was
carriedout by trapping them in Tenax
TATM coated stainless steel desorbing columns. The crude antibiotics of
the effective BCAs were analyzed for the detection of active biomolecules
responsible for the suppression of pathogens through GC-MS (GC Clarus 500
Perkin Elmer). Using database searches on
the NIST version 2005 MS data library and comparing the spectrum obtained
through GC/MS, the compounds present in the crude sample were identified.
Screening of antifungal metabolite against L. theobromae
The diluted metabolite from the
antagonists was further tested for their antagonistic property against the
fungal pathogen using the agar well diffusion assay. 25, 50, 75 and 100 mL crude metabolites of the BCAs were placed inside the
PDA wells. The mycelia of the test pathogen were inoculated in with an agar
plug of 9 mm dia. in each plate in the center of the Petridish.
The percentage inhibition was studied after 5 days by calculating the
inhibition percentage.
Statistical analysis
The data observed from the above
study were analysed using the statistical tool for
agricultural Research (STAR 2.0.1). For comparing the treatment means Tukey’s
honest significance difference test was used, and the significant level of
treatment was calculated by the magnitude of the F value (P < 0.05). Plant growth parameters were calculated by the mean ±
standard deviation using GraphPad Prism (v. 6.0) and further comparisons was
conducted using DMRT at P < 0.05
(XLSTAT).
Results
Survey, isolation and selection of virulent isolate
A survey was conducted in various
districts of Tamil Nadu to assess the occurrence of L. theobromae in eriophyid infested
coconut. The results of the report revealed that the incidence of L. theobromae
was varied from 30.60 to 88.12 (PDI) in all districts of Tamil Nadu. Maximum
occurrence of the disease was recorded in Vizhupuram
(PDI- 88.12), followed by Attur, Salem district (Fig.
1).
The organism grown on PDA and produced a light gray, fluffy and aerial mycelium. The culture became
dark in colour in advanced stages. Mycelium was inundated
or superficial, branched, septate and brown. Dark brown flask shaped,
ostiolate pycnidia appeared in
7-day-old cultures. The ostiole was circular and arranged at the apex of an elongated neck through which pycnidiospore
extruded. Pycnidia varied in size, 125–180 µm ×
80–145 µm. Pycnidiospores
were at first hyaline globose to oval
and unicellular, but became brown and 1-septate with
age and measured 20.5–30.0 µm ×
11.0–13.5 µm. Based on
characteristics of the pycnidia and pycnidiospores,
the fungus was identified as Botryodiplodia theobromae
(Pat) syn. Lasiodiplodia theobromae
Pat. Griffon & Maubl (Mullen et al. 1991; Woodward et al.
2005). Maximum No. of pycnidia (90 per plate) with more matured pycnidiospores (65.3) were produced by the isolate L26. The
isolate L26 produced highest numbers of pycnidiospore
within a short period and it would have the capacity to infect healthy nuts.
From this study the isolate L26 was selected for entire studies (Fig. 1). From
a total of thirty isolates collected from different places in Tamil Nadu, all
the isolates confirmed as L. theobromae by PCR technique (Fig. 2). All the thirty
could get an amplification size of approximately 560 bp.
In vitro effect of BCAs on L26 isolate
The antagonistic assay was
performed using dual plate technique and the radial mycelial growth of the
pathogen was monitored. The BCAs, T. asperellum, B. subtilis and S. rochei inhibited the mycelial growth
of L26 and the percent inhibitions
were 67.11, 58.49 and 57.48 respectively, the clear
inhibition zone exhibited the antagonistic activity after four days of
incubation (Fig. 3). Likewise, other antagonist also inhibited the mycelial growth of the
pathogen from 37 to 46%. The spore inhibition percentage also increased from
73.75 to 58.95 (Table 1 and 2). Based
on this antagonistic potential the three antagonists were taken for further
study. The antagonists also caused extensive hyphal coiling, lysis and thinning
respectively and less dense hyphal network compared to control. Light microscopy of the fungal
mycelia revealed that distortions, damages and also shrinkage of L26 isolate in
the treated plates. In addition, the hyphae were parasitized by spores of the
antagonist. In control plate they were no such changes were detected (Fig. 4).
Table 1: BCAs with their accession
Numbers
Isolate No. |
BCAs |
Accession number deposited
in NCBI |
1 |
Streptomyces rochei |
MT122809 |
2 |
T. asperilloides |
Y848322 |
3 |
T. koningeopsis |
MF423101 |
4 |
T. harzianum |
KX533990 |
5 |
T. asperellum |
KX533985 |
6 |
B. subtilis |
KF718836 |
7 |
B. amyloliquefaciens |
WODE00000000 |
8 |
B. lichiniformis |
MG241257 |
9 |
B. megatherium |
CP032527.2 |
Table 2: In vitro effect of antagonists against
radial mycelial growth and spore germination of L26
Sr. No. |
Antagonists
|
Radial mycelial growth |
% growth inhibition over
control |
(%) spore germination * |
(%) spore inhibition over
control |
1 |
Trichoderma asperellum |
28.30 (38.22)a |
67.11 |
23.25 (35.20)a |
73.75 |
2 |
Bacillus subtilis |
36.00 (42.70)b |
58.49 |
32.70 (46.66)b |
63.45 |
3 |
Streptomyces rochei |
36.90 (43.22)c |
57.48 |
36.83 (40.80)c |
58.95 |
4 |
Bacillus amyloliquefaciens |
47.90 (43.79)c |
46.36 |
46.11 (42.77)cde |
49.73 |
5 |
Trichoderma harzianum |
49.80 (44.88)cd |
44.23 |
53.47 (46.99)cd |
41.71 |
6 |
Bacillus lichiniformis |
51.10 (45.63)cd |
48.78 |
48.85 (44.34)de |
46.75 |
7 |
Trichoderma koningeopsis |
52.00 (46.14)cd |
41.77 |
49.87 (43.18)cde |
45.63 |
8 |
Trichoderma asperilloides |
54.00 (47.29)cd |
39.53 |
54.52 (47.60)cde |
40.56 |
9 |
Bacillus megatherium |
55.80 (48.33)cd |
37.51 |
52.89 (46.66)e |
42.34 |
10 |
control |
89.30 (71.47)e |
|
91.73 (73.30)de |
|
SEd
(0.01) |
|
|
4.23 |
|
|
*Mean of three replications.
Values in parantheses are arc sine transformed values.
In a row, means
followed by a common letter are not significantly different at 5% level by DMRT
Fig. 1: Prevalence and morphological
variation of L. theobromae
Antifungal volatiles test-inverted bioassay
Antifungal volatiles from the
antagonists were studied against L26 using inverted bioassay method. It was
noticed that the presence of volatile compounds adversely affect the mycelial
growth of L26 on the 3rd day. There was reduction in percent
mycelial growth (59.61, 53.20, 47.03%) in all BCAs compared to control. As
support to these results, there was no pycnidia formation in all three test
plates where as in control there was increased or more number of pycnidia on
day of seventh. The S. rochei explored plates had sparse mycelial growth
without pycnidia production and there was no inhibition percentage in mycelial
growth but reduction in spore formation. The effectiveness of the volatile
compounds on the mycelial growth and pycnidia production of the test pathogen
proved the suppressing ability of the antagonist under in vitro (Fig. 5).
Volatilome pattern associated with antagonists with L26 isolate
The vo
Fig. 2: Agarose gel electrophoresis showing ITS1,
5.8S I and ITS4 ribosomal DNA PCR product of L. theobromae
Fig.
3: In
vitro effect of T. asperellum, B. subtilis and
S. rochei against L. theobromae
1: T. asperellum vs. L26; 2: B. subtilis vs. L26; 3: S. rochei
vs. L26; 4: Control (L26)
Fig.
4:
Light microscopic image of L26 taken at the interaction zone with the
antagonists
a. Image of L26
without interaction; b. Hyphal
septum malformation and branch deformation during the interaction with
antagonists
Fig. 5: Effect of volatile metabolites of
antagonists on pycnidia formation of L26 isolate
1: T. asperellum vs. L26; 2: B. subtilis vs. L26; 3: S. rochei
vs. L26; 4: Control (L26)
latile compounds
from the
antagonists had an inhibitory effect on L26. Totally, 138 mVOCs were identified, with typical mass spectra and a
broad range of molecular weights, ranging 51–492 g mol-1. The
deconvoluted mVOCs belonged to eighteen classes, the
most dominant class is the Benzene derivatives, followed by Heterocyclic organic
compound, Terpenoid and Pyrone derivatives and the trend is strong in the
interaction of antagonists (Tv, Bs, Sr), with L26 isolate.
GC-MS results revealed that antagonists
(Tv) emitted terpenoid compounds (β-caryophyllene
(7.667%) with peak areas, followed by isoamyl alcohol (3-methyl-1-butanol
(15.764%) and benzene derivatives (1, 2-benzenedicarboxylic acid (22.877%),
2-butoxy-2-oxoethyl butyl ester (24.655%). The antagonists B. subtilis also emitted benzene derivatives (benzothiazole
(5.192%), cyclic lipopeptides (surfactin A
(9.687%), saturated hydrocarbons (alkanes (4.064%) and S. rochei emitted few organic compounds viz., hexahydro-pyrrolo[1,2-a]
pyrazine-1,4-dione (7.655%), 2-piperidinone (22.876%),
hexahydro-3-(phenylmethyl (15.664%). The pathogen L. theobromae, produces, cyclohexenes and
cyclohexenones (4R, 5R)-4,5-dihydroxy-3-methylcyclohex-2-enone,
theobroxide), jasmonates
(methyl jasmonate, (11R)- 11-hydroxy-jasmonic acid). The intensity of peaks of these
compounds increased during the interaction with antagonists with L. theobromae the peak intensity for terpenoids (β-caryophyllene) was the
predominant class, followed by phenols (Ketoconazole) and Heterocyclic
organic compound (2-piperidine) were increased on their interaction (Fig. 6a–e).
However, the peak intensity of 5.19% corresponding to pyrrolo-quinoline derivatives
decreased in the interaction of L26 with antagonists (Fig. 8).
Screening of antifungal metabolite against L. theobromae
In agar well diffusion method the metabolites of the three antagonists,
showed good antifungal activity against the test pathogen. The dilution from 25 µL to 75 µL of antimicrobial
extract was poured in three replications with a control. The 25 and 50 µL of the crude extract from T. asperellum inhibited 85.12 and 97.32%
of mycelial growth of L26. There is absolute reduction in mycelial growth at 75 µL concentration. The extract from B. subtilis
at the concentration of 25 and 50 µL showed 86.03 and 95.93 percent mycelial growth. The antagonist S.
rochei inhibited the mycelia of the pathogen at
different concentrations of 25, 50 and 75 µL were 84.09, 93.38 and 100%. At the concentration of 75
µL of the extract showed that, complete inhibition in treated compared to control. There was no mycelial growth was
observed in 75 µL of crude
extract treated plates inoculated with L26 (Fig. 7).
Discussion
For many years, the
etiology of the destructive symptom in coconut was deserted by researchers and
in spite of the value of this disease, only L.
theobromae had been reported as a pathogen to
coconut. It causes heavy losses in coconut being a very destructive fungal
pathogen worldwide. For the proper identification, distribution, yield loss of
the pathogen, the survey was conducted and isolates were collected and
validated. Based on the survey report all the isolates were causing the same
symptom in coconut with a difference in level of incidence. The pathogen was
identified on morphological and molecular basis and identified as L. theobromae.
The most frequently described morphological characteristics include mycelial
growth, pycnidia and pycnidiospore production
(Machado et al. 2014; Linaldeddu et al.
2015). Similarly, Ashokkumar et al.
(2018) reported that all the isolates
showed variation within the morphological characters and the pycnidiospore estimate changes from 14.3 × 7.69 μm (LT-CL2) to 25.59 × 13.31
μm
(LT-CL5). Latha et
al. (2013) reported that, isolates
from Coimbatore (TNAU), showed dark
grey colonies and found to be more virulent
as it produced maximum pycnidia (63) in 90 mm Petridish.
The virulent was distinguished
based on the pathogenicity and production of pycnidiospores under in vitro.
Initially nine BCAs were tested against the
fungus, based on the inhibition percentage the three BCAs were selected and
they were having highest percentage of inhibition in dual plate assay. It is
known that secondary metabolite production, could be strongly influenced the
culture growth of the pathogen (Miao et
al. 2006). The Different volatile organic compounds from Trichoderma
have been profiled with antimicrobial properties and it is specific to
different species and strains of Trichoderma (Dennis and
Webster 1971). A Bacillus
spp. TN79 derived from Thua Nao, suppress the growth of L. theobromae (Chukeatirote
et al. 2018). Liu et al. (2019) reported that, S. sclerotiorum
and R. solani isolated from soybean root were
inhibited by Streptomyces spp. NEAU-S7GS2. Furthermore, Srivastava et al. (2015) described that mycelial
formation and percent disease progress of S. sclerotiorum
was suppressed with S.
rochei strain SM3 at an approximate rate
of 74%. Similarly, Yu et al. (2020) and Martinez et al. (2020) reported that S. triticiradicis
and S. lydicus
caused inhibitory effects against pathogenic
fungi from 30 to 63%. Hydrolytic enzymes such as chitinases or glucanases or proteases and may be the production of
antibiotics against the fungal cell wall of L.
theobromae was the cause for the zone of
inhibition formed by the ultrafiltered crude extracts of BCAs in light
microscopic observations (Jain and Jain 2007; Yan et al. 2008; Oskay 2009).
Fig. 7: Effect of extracted
metabolites of the antagonists on the radial growth of L26
Fig. 8: Compound classes present in axenic
and co-culture
The volatile compounds have an
indirect effect, without any contact with between antagonists and pathogen. In
this study, there was reduction in percent mycelial growth compared to control
and the hyphae regain their development once the antagonists were removed from their atmosphere.
This evidence demonstrates that volatile compounds from BCAs are having
fungicidal effect and similar results were obtained for sclerotia germination,
in the presence of T. reesi,
T. harzianum, and T. longibrachiatum EF5 the sclerotial germination were reduced in S. rolfsii isolates (Sridharan et al. 2020).
In our study, we identified mVOCs with antimicrobial properties from axenic culture of T. asperellum
through GC-MS-TD such as, peptaibols (aspereline), limonene; β-eudesmol and
1, 3-octadiene. Wilkins et al.
(2003) reported that the metabolite delivered
by T. viride
(2-propanol, 3-methyl furan, 1-pentanol, 2-hexanone), T. atroviride (pentanones, octanones, etc.),
T. harzianum
(cyclohexane, alcohols, esters etc.)
showed hindrance in growth of pathogens and 141 compounds
counting a few obscure sesquiterpenes, diterpenes and tetraterpenes from Trichoderma spp. (Lee et al. 2016). The VOCs emitted from B. subtilis was 2, 4-di-tert-butylphenol,
also coincides with the results of Ongena and Jacques
(2008); Wang et al. (2007) and Yoshida et
al. (2001). The large
amount antimicrobial activity is attributed to iturin
and fengycin was reported by Robacker et al. (1998);
Kai et al.
(2009) and Caulier et al.
(2019). From Bacillus
spp. Streptomyces spp. are regarded as noteworthy sources in generation of secondary metabolites
and these antimicrobial compounds could play parts in securing plants against distinctive
pathogens (Miyada et
al. 2017). In our study, S. rochei emitted 2-piperidinone and pyrrolo[1,2-a]
pyrazine-1,4-dione and few other VOCs. From our investigation,
it is observed that the pathogen L. theobromae emitted
cyclohexenes and cyclohexenones (4R,5R)-4,5-Dihydroxy-3-methylcyclohex-2-enone,
Theobroxide), jasmonates
(Methyl jasmonate, (11R)-11-Hydroxy-jasmonic corrosive) and leads to the restraint of the defense pathway of the plant host, encouraging the disease process (Tsukada et al.
2010; Chanclud and Morel 2016). Few
known secondary metabolites, such as jasmonic acid and 3-ICA,
phytotoxin scytalone were identified
from Lasiodiplodia
species (Felix et al. 2018).
Remarkably, our study suggests that several
biochemical pathways be drawn in in the construction of VOC in microbes while
they relate with one another. These include cis-calamenene
related sesquiterpenoids biosynthesis, phenolic malonylglucosides
biosynthesis with Terpenophenolic Biosynthesis
pathways.
During the interaction experiments, we
pragmatic a boost in the expression of volatile compounds. This upregulation of mVOCs might be owed to
the existence of diverse genus in the similar culture media. Signal molecules,
accountable for the intra- and interspecies communication, are activated
throughout these relations. This activation may perhaps be a consequence of
more than a few volatile and non-volatile metabolites unconfined during the
early development stage by together the microbes. Those metabolites were formed
from any of the microorganism’s viz., BCAs, L. theobromae
and this verdict was in lineup with the result of Karuppiah and coworkers (2019). Toffolatti
et al. (2021) described that terpenoids act as specific or universal
pathogen inhibitors. Similarly, in our study
during the interaction of BCAs with L26, production of the class terpenenoids was found to be increased and this is one of
the responses to assail in several plant-pathogen binomials. As well, phenols (Ketoconazole), act as
signaling cursor, organization of arbuscular mycorrhizal symbioses and that can
act as agents in plant defense (Mandal et
al. 2010) and it is evident from our study during the interaction.
Mahmoud and his coworkers also obvious our results that 2-piperidine, a heterocyclic organic compound disrupt the interaction shut between enteric pathogens
and these derivatives have a good inhibition capacity against most
tested pathogenic bacterial and fungal species due to their potential
antimicrobial and fungicidal properties (Mahmoud et al. 2018). Pyrrolo-quinoline derivatives
level is reduced in the coculturing experiments and in assessing redox cycling
these derivatives has more than 100 times efficient than polyphenolic
compounds, ascorbic acid, isoflavonoids potentials (Stites et al.
2000) and this might be a result of a few unstable and non-volatile metabolites discharged
amid the beginning stage by both the microorganisms. Those metabolites were created from either of the microorganisms viz., T.
asperellum, B.
subtilis, S. rochei, L. theobromae nor interaction of those
and this finding was in line with the findings of related proteins,
secondary metabolites and plant growth
promoting compounds to essentially
improve the plant
development and assurance against
plant pathogens. However, no reports have illustrated the effects of
co-cultivating the two most agriculturally important bacteria and fungi along
with the pathogen (T. asperellum, B.
subtilis, S. rochei and L. theobromae)
on the metabolite production. In this paper, we have tended
to this address a few co-cultivation
technologies come about in improved
action for various secondary metabolites, but frequently
not for all.
Conclusion
Pycnidial producing fungi, L. theobromae proved to be a virulent
pathogen in coconut and act as secondary pathogen next to eriophyid mite. The
BCAs, proved to be effective against L. theobromae by producing mVOCs
by inhibiting the pycnidiospore production. The
compounds emitted from these BCAs can be deliberate further for additional
properties, like growth promotion, medicinal properties, nutrient mobilization etc. Consequently, further
it needs to be confirmed by
developing a formulation from these biomolecules along with BCAs for more
antimicrobial activity. Hence, an active bundling with these substances may be
a great alternative to manage
the incidence in coconut caused by the dreadful L. theobromae.
Further, the scale-up of this co-cultivation technology within the fermentor and field study will encourage the use in farming sector.
We greatly thank the colleagues of the department of
Plant Pathology, Tamil Nadu Agricultural University, Coimbatore for their
support and providing access to the facilities needed for preparation of all
the cultures. Additionally, we thank the colleagues from department of
Microbiology, for their support and providing access to the facilities needed
for the microbiological assays using GCMS.
AS, MR and MS conceived the study. AS performed the
experiments and analyzed the data. MR coordinated the experiments and helped to
draft the manuscript. MS coordinated the experiments related to eriophyid mite.
AS, MR and MS drafted the manuscript and wrote R scripts and analyzed the data.
All the authors were contributed to the chemical analysis and authentic
reference standard measurements. All authors have read and approved the
manuscript before submission.
The authors declare that the research was conducted in
the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
All datasets presented in this study are included in the
article/supplementary material
Not applicable
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