DISCUSSION

This study explored the endosymbiotic microbes in plant as well as insect system and
evaluated molecular and biochemicalnature of their interaction with their host.The study
provides an insight to the secondary metabolites, metabolic pathways and genes the
endosymbionts, that help in the production of bioactive compounds for ecological,
therapeutical and bioremediation purposes. Thus, we studied the endosymbiotic microbes
ofthree medicinal plants (A. indica, C. longaand M. charantia), various ant species
(includingOecophyllasmaragdina, Camponotusfloridanus, Solenopsis, Anoplolepisgracilipes
and Camponotusfloridanus) and various niches(focusing on the PAH degrading microbes).In
summary, about 271 different genera of microbes as endosymbionts were studiedamong
which bacteria: fungi were in ratio 43.5:56.5 ratio. Most abundant was genus Fusarium with
7% of total fungal species, followed by pseudomonas, with 4.8% of total bacterial species.
The most abundant phylum in medicinal plants were Ascomycota, represented by
Aspergillus, Penicillium, Trichoderma, Alternaria, Phoma, Verticillium, Cladosporium,
Xylaria, Phomopsis and Diaporthe. It has been reported that Ascomycota fungi have higher
species diversity due to their faster evolutionary rate and adaptability [0]. This may explain our result
that the fungal communities were predominated by Ascomycota. Among bacteria, most abundant
family was Proteobacteria followed by Firmicutes (represented by Pseudomonas and Bacillus
respectively). This can be explained by Proteobacteria accounting for most of the isolates
possessing siderophore production, phosphate solubilization, IAA-like indole derivatives production,
HCN production and ß-glucosidase activity, respectively. Firmicutes are also enriched in isolates
showing ß-glucosidase activity, siderophore production, and IAA production.Phosphate solubilization
activity is also shown by Proteobacteria[1]

In Neem, a total of 61 microbial species belonging to 28 genera were studied. The most
dominant genus was Fusarium (10%). The rich diversity of Fusarium species as endophytes in
different tissues of both the plants may be due to germination of more number of spores of this fungus
due to favourable environmental condition.[5] . 74.4% of microbes were bacteria and 25.4% were
fungi.

In Turmeric, a total of 61 microbial species were studied. The most dominant genus was
Bacillus (9.83%). In Bitter gourd, a total of 66 microbes were studied. The most dominant
genus was Fusarium (13.8%), followed by Arthrinium (7.7%).

The most dominating genus amongst bacteria was Pseudomonas with 8.13% bacterial strains.
This might be explained by production good quantity of siderophore, IAA, and solubilized
inorganic phosphate by Pseudomonas spp. It also enhances the growth and antioxidant properties of
the host plant[6]. Amongst fungi was Aspergillus with 25% fungal strains. 22.1% of bacteria
were gram positive, 17.1% of bacteria were gram negative with undefined locations and the
rest were eitherGram-Negative and soil, water microbe 2.9% or Gram Negative and soil
microbe 2.9% or Gram-positive and soil, water endophyte 2.9% or Gram-negative bacteria
and marine microbe 2.9% or Gram-positive and soil microbe 8.6% or Gram-negative and
marine microbe 5.7%. The percentage of lignolytic fungi was 66.7%. In bacteria,
Deoxygenase was the most recurring enzyme. The catechol dioxygenases serve as part of
nature’s strategy for degrading aromatic molecules in the Environment. They are found in the soil
bacteria and involved in the transformation of aromatic precursors into aliphatic products. The
intradiol cleaving enzymes utilize Fe(III), while the extradiol cleaving enzymes utilize Fe(II) and Mn(II)
[7], followed by dehydrogenase. Laccase was the most recurring enzyme in fungal
species.Laccases are capable of catalyzing the oxidation of ortho and paradiphenols, aminophenols,
polyphenols, polyamines, lignins, and aryl diamines as well as some inorganic ions. Laccases not only
oxidize phenolic and methoxyphenolic acids, but also decarboxylate them and attack their methoxy
groups (demethylation) [8][9][10]. 60% of bacterial species showed complete metabolism,
31.42% showed either co-metabolism or complete metabolism, and only 5.71% showed co-
metabolism.

In ants and in microbes isolated from various niches also, the most abundant family was
Proteobacteria followed by Rhizobium and Burkholderia, Enterobacter, Hemophillus,
Pseudomonas, Vibrio, Cycloclasticus, Aeromonas, and Alcanivorax. Among ants, a total of
36 microbes from 27 different taxa (12 genera, 15 species) were studied. 100% of them were
bacteria. The most dominant genus was Wolbachia (8.6%), followed by Pseudomonas,
Rhizobium and Arsenophonus (5.7%).Wolbachia exhibited use in the regulation of cell
growth, differentiation, diverse cell functions, and apoptosis[2][3]. It also produces potent
immunostimulants.[4]

It is possible that an endophyte might be a causal agent of a plant or animal disease but still
produce compounds with therapeutic properties. For example, Cladosporiumcladosporioides
isolated from Neem causes rot of red wine grapevines. It can occasionally cause pulmonary
and cutaneous phaeohyphomycosis and it has been isolated from cerebrospinal fluid in an
immunocompromised patient. This species can trigger asthmatic reactions due to the presence
of allergens and beta-glucans on its spore surface. But, it has been shown that when isolated
from Taxus media, it produces taxol. In addition, p-Methylbenzoic acid, found in
C.cladosporioides, facilitates 1,5-benzodiazepine synthesis. Benzodiazepines are commonly
used as anti-anxiety, anticonvulsant, anti-depressant, anti-inflammatory, analgesic, and
sedative agents (Tsoleridis et al., 2008). Benzodiazepines have a range of actions including
antiulcer, antileukaemics, vasopressin antagonists, antiplatelet and endothelial antagonists
and skeletal muscle joint pain relief (Kumar and Joshi 2007; Aasth et al. 2013).

Similarly, Aspergillus versicolor was isolated from Turmeric. Aspergillus versicolor is the
major producer of sterigmatocystin, a carcinogen which is a precursor of the aflatoxins (Cole
and Cox, 1981)..But, many metabolites produced by A. versicolor exhibit antibacterial,
fungicidal, insecticidal, and cytotoxic properties. For example, a sesquiterpenoidnitrobenzoyl
ester isolated from hyphae have been shown to be potent inhibitor of human breast and colon
cancer cell lines. Other extracted compounds that are cytotoxic towards cancer cells include
xanthones, fellutamides, and anthraquinones. Anthraquinone tends to be yellowish in color,
and like other pigment molecules, A.versicolor produces it frequently . Other tests of the
fungus have shown different metabolites with bacterial involvement, such as M.
Tuberculosis, yeasts similar to C. Albicans. Aspergillomarasmine A was reported to inhibit
two carbapenemase antibiotic resistance proteins in bacteria.  A. Versicolor can  also
effectively decolor the large concentrations of Remazol Blue reactive dye and heavy metal in
growth medium molasses.

Penicillium, an important fungal genera reported in Neem and Turmeric, plants is known for
its ability to produce a range of bioactive secondary metabolites with well-proven biological
activities (Petit et al., 2009; Gao et al., 2010; Zhelifonova et al., 2010).

From bitter gourd, two species belonging to genus Chaetomium madrasense and brasiliense
were isolated. C. madrasense is considered to be a rather common species (Arx et al. 1986).
The obtained results suggested that the C. madrasense had the potentiality as a source of
strong natural and safe antioxidants safe for application in the food and cosmetics industries.
Chaetomadrasins A (1) and B (2)were isolated from the solid-state fermented culture of
desert soil-derived Chaetomium madrasense 375. Compounds 1 and 2 displayed moderate
cytotoxicity against HepG2 human hepatocellular carcinoma cells. The fungus Chaetomium
brasiliense is one of the 22 Chaetomium species that have been found in Thailand.
Mollicellins K-N (1-4), and six known depsidones, mollicellins B (5), C (6), E (7), F (8), H
(9), and J (10) were extracted from C. brasiliense. Only 1 exhibited antimycobacterial
activity against Mycobacterium tuberculosis and antifungal activity against Candida albicans
using in vitro assays. In addition, 1-10 showed cytotoxicity against the KB, BC1, NCI-H187,
and five cholangiocarcinoma cell lines.
Interestingly, similar genes and pathways were found among those of the bioactive
compounds and endophytes fumigaclavine A dimethylallyltransferase and Prenyltransferases in
Aspergillus sp. , Ochratoxin A biosynthesis, Patulin biosynthesis in Penicillium sp., FusA (fusarin
C synthetase), FUB1 (fusaric acid synthase) in Fusarium monoliforme. AZAI (azaphilone
biosynthesis cytochrome P450 monooxygenase) in Chaetomium globosum.

In ants, Wolbachia and Wolbachia arsenophonus had same function of influencing sex ratio
while in Formica cinereahad possible effects on social life. Additionally,11.1% of microbes
may have had possible effects on social life and queen-worker sex allocation conflicts. Rest
were involved in functions like reproductive development and nutritional upgrading
(Blochmannia floridanus in Camponotus floridanus), amino acid supply (Oecophyllibacter
saccharovorans in Oecophylla smaragdina) and anti- microbial compound synthesis
( Pseudonocardia and Streptomyces in Atta sp.). Interestingly, Serratia symbiotica had three
different functions in four different ant species

Polycyclic aromatic hydrocarbons (PAHs) are carcinogenic compounds with two or more
fused benzene rings that originate from both natural sources and anthropogenic activities. A
total of 16 PAHs have been listed as toxic pollutants by the US EPA (Perelo, 2010) .They are
highly hydrophobic in nature and tend to adsorb onto the surface of soil (or sediments in a
marine environment).Phenanthrene, naphthalene and anthracene are seen to be the most
widespread and easily atrophied. 126 unique genes were studied. Most dominant gene
wasnidA (12%)followed by pdoA (4.8%), nidD (4%), nidB (4%) and alkB (4%).

The key enzymes which played a role in the process were oxygenase enzymes in microbes
and cytochrome P450 enzymes in fungi despite it being Ligninolytic and Non- Ligninolytic,
this can be catabolised through co-metabolism or as a sole carbon source.

Bacteria play an important role in the removal of polycyclic aromatic hydrocarbons (PAHs)
from polluted environments. In marine environments, Cycloclasticus is one of the most
prevalent PAH-degrading bacterial genera. Naphthalene degrading bacteria are ubiquitous in
nature and there are enormous numbers of reports documenting the bacterial degradation of
naphthalene including the elucidation of the biochemical pathways, enzymatic mechanisms
and genetic regulations (Cerniglia, 1992; Peng et al., 2008; Seo et al., 2009; Lu et al., 2011;
Mallick et al., 2011)Principally, bacteria favor aerobic conditions for degradation of PAHs
via oxygenase-mediated metabolism (involving either monooxygenase or dioxygenase
enzymes). Usually, the first step in the aerobic bacterial degradation of PAHs is the
hydroxylation of an aromatic ring via a dioxygenase, with the formation of a cis-dihydrodiol,
which gets rearomatized to a diol intermediate by the action of a dehydrogenase. The
biodegradation of PAHs by fungi has been studied extensively in last several years and
numerous fungal species have been reported to metabolize different PAHs (Cerniglia, 1992;
Cerniglia and Sutherland, 2010). Most fungi cannot use PAHs as sole sources of carbon and
energy; however, they may co-metabolize PAHs to a wide variety of oxidized products and
sometimes to CO2. Bacterial PAHs degradation mainly involves dioxygenase enzymes and
partially monooxygenase mediated reactions and the same is valid for algae. For example, the
extent of dioxygenase vis-à-vis monooxygenasecatalyzed transformation of naphthalene by
Mycobacterium sp. was found to be in the ratio of around 25:1 (Kelley et al., 1990). On the
other hand, fungal PAHs degradation mainly involves monooxygenase enzymes (Cerniglia
and Sutherland, 2010) (and the references therein). However, the transformation of PAHs by
fungi involves several enzymatic pathways and depends on the particular species and growth
conditions. The fungi involved in PAHs biodegradation are mainly of two types- ligninolytic
fungi or white-rot fungi (they have the ability to produce enzymes including lignin
peroxidase (LiP), manganese peroxidase (MnP) and laccases to degrade the lignin in wood)
and non-ligninolytic fungi (those who do not produce peroxidases or laccases but instead
produce cytochrome P450 monooxygenase like enzymes) (Hofrichter, 2002; Tortella et al.,
2005; Cerniglia and Sutherland, 2010; Li et al., 2010).
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