Evaluating Oregano (Origanum vulgare) Extract Fungicidal Activity Against Banana

Bunchy Top Virus in Bananas (Musa acuminata) caused by banana aphids (Pentalonia
nigronervosa)
Panabo City National High school
Research Proponent-Princess Jamica Libranza
Research Teacher- Cindy S. Valderama
Grade10-Andromeda
Research 10
___________________________________________________________________________

CHAPTER 1 - INTRODUCTION

Background of the study

One of the biggest problems in Banana production is controlling the quality of every banana,
they could produce. Maintaining the quality from the planting down to exporting process.
Various disease may damage not only the quality of the product but also the quantity. Banana
aphids (Pentalonia nigronervosa) are small brown to almost black oval shaped aphids. They
create colonies that can be found on the pseudostem of banana plants under the leaf between
the bunch bracts and the branch stalk. They can also create colonies in the unfurled top leaves
of young plants. These pests could cause small but frequent damage in the plant.

The banana aphids (Pentalonia nigronervosa) are the insect vectors of Banana Bunchy Top
Virus (BBTV), the viral cause of banana bunchy top disease (BBTD). The aphids can acquire
and transmit the banana virus during their feeding on infected banana cells.

Oregano Origanum vulgare a type of leaf that came from the family of Lamiaceae. The
oregano plant is very rich in flavour and odour1. Other than the use of oregano as an
insecticide, oregano plants also play a big role in the evolution of the food industry. In some
countries the name of the oregano plant means “Joy of the mountain” because of its multiple
functions. The focus is on how the plant will help the environment as an insecticide agent.
These plants contain chemical compounds that can help to stop the growing population of
flies musca domestica. Oregano oil has chemical compounds such as carvacrol, β-fenchyl
alcohol, thymol, and γ-terpinene.2 Oregano is one of the most traded and consumed medicinal
plants worldwide.

1
2
Rationale and significance
The main goal of the study is to create a sustainable and low cost pesticide to help poor
banana growers. The results of this study is not focused to the positive results but to the other
room of choice.

Conceptual /Theoretical Framework

Oregano is usually used for herbal treatments and many other uses. In order to make use of
the oregano plant, the researcher will use its natural pesticidal components.

The Oregano leaves will be extracted using the hydrodistillation method.

Review of Related Literature

Banana Aphids

Probably native to Southeast Asia, the banana aphid is present just about everywhere banana
is grown through the world. It occurs in tropical Africa, Atlantic Islands, Australia,
California, Central America, Cook Is., Egypt, Fiji, Florida, Hawaii, Kiribati, India, Indonesia,
Malaysia, Madagascar, Marshall, Is., Mauritius, Mexico, Micronesia, the Middle East,
Mozambique, New South Wales, Papua New Guinea, the Philippines, Reunion, Samoa,
northern South America, Taiwan, Tokelau, Tonga, Tuvalu, Vanuatu, Wallis Is., and much of
the West Indies (Waterhouse, 1989).

The banana aphid was first recorded in the state in 1924 from Honolulu, but was previously
known in the Islands as an undetermined species (Zimmerman, 1948). Although known to be
on Oahu, Hawaii, and Kauai, it wasnÕt recorded on Maui, until 1989 (Heu, unpublished
data).

The banana aphid (Pentalonia nigronervosa) has a worldwide distribution, and in 1925 was
shown to be a vector of BBTV. It remains the only known insect vector of this virus. Hosts of
the aphid include species in the family Musaceae and several related families, including
Araceae, Heliconiaceae, Strelitzeaceae, and Zingiberaceae, though the aphid shows a degree
of host preference and can be difficult to transfer between host species. On banana, they
commonly colonize the base of the pseudostem at soil level and for several centimeters below
the Transmission by P. nigronervosa is of the persistent, circulative, nonpropagative type, and
individuals from areas where the virus is endemic and from where it is absent, both transmit
the virus with equal efficiency. There is a minimum acquisition access period of 4 h, a
minimum latent period of a few hours, and a minimum inoculation access period of 15 min.
Aphids retain infectivity, after removal from a virus source, for at least 20 days and probably
for life. Both nymphs and adults can acquire the virus, though more efficiently by the former,
and reported transmission rates for individual aphids are in the range 46–67%. There is no
evidence for transmission of BBTV to parthenogenetic offspring or for replication of the
virus in the aphid.

The banana aphid, Pentalonia nigronervosa Coquerel, is present worldwide where banana
(Musa spp.) is grown. It is the vector of Banana bunchy top virus (Nanoviridae, Babuvirus),
the etiological agent of banana bunchy top disease, currently the most important constraint
for banana producers in Hawaii. P. nigronervosa is not well studied, and effects of
temperature on its growth and reproduction are unknown. We studied the longevity and
fecundity of one clone of banana aphid on different types of plant materials to determine an
effective method to study the insect in the laboratory. We found that insects performed better
unconfined on plantlets, followed by leaf midrib cuttings. We also conducted complete life
table studies with P. nigronervosa on banana leaf midrib cuttings at 20, 25, and 30°C, with a
photoperiod of 12:12. Intrinsic rate of increase (r), net reproductive rate (R 0), doubling time
(DT), nymphal mortality, and mean offspring per female all showed maximal rates at 25°C.
Population growth was studied on whole banana plantlets as well, and growth rates were also
highest at 25°C. We found r to be greater when aphids were reared on intact banana plantlets
than on cuttings. Our results show the importance of comparing insect rearing methods for
studies such as life tables.

Banana bunchy top virus

BBTV is known to be the most destructive virus disease of banana in the Philippines with no
known cure. The virus is carried and spread by aphids scientifically called Pentalonia
nigronervosa. The infective aphids transmit the virus to a banana plant by feeding on it. Its
infection causes stunting and leaf malformation leading to the host’s premature death.

BBTV is also efficiently transmitted in vegetative planting material, both conventional

corms, corm pieces (bits) and suckers, and through micropropagation. All meristems from an
infected corm will eventually become infected.soil surface, beneath the outer leaf sheaths and
newly emerging suckers.

Banana bunchy top virus (BBTV, genus Babuvirus, family Nanoviridae) causes the most
destructive viral disease of banana worldwide, but is restricted to some countries in Africa
and the Asia-Pacific region. Hosts of the virus are found only in the plant family Musaceae,
and it is transmitted through vegetative planting material and in a circulative, nonpropagative
manner by its sole vector the banana aphid (Pentalonia nigronervosa). In banana, disease
symptoms include stunting, leaf chlorosis, and rosetting, and a cessation of fruiting, and no
natural source of immunity is known. Histological effects and virus replication are restricted
to the phloem tissue. The virus has isometric virions, 18–20 nm in diameter, separately
encapsidating each of the six integral circular single-stranded DNA (ssDNA) genome
components. All components are monocistronic, except for DNA-R, which encodes the
master replication initiation protein, and a smaller internal open reading frame (ORF) of
unknown function. Replication is via a rolling-circle mechanism, and the genome
components share two conserved areas in their respective untranslated regions; the stem loop
common region that contains the origin of replication and the major common region which
binds a heterogeneous population of DNA primers, around 80 nt in length, that prime
complementary strand synthesis.

Banana aphids can be found on the pseudostem, youngest unfurled leaves

and on the underside of the old leaves of Abaca (Lalusin and Villavecenccio,
2016). They are mostly found on the petiole of abaca leaves and outside of
the furled young leaf of the plant. Based on vector transmission of the virus to
the plants, it has been reported that ABTV and BBTV can be acquired by the
plants within two hours of an ‘acquisition feeding period’ (Bajet and Magnaye
2002) with a single aphid, although infection is more pronounced if a greater
density of aphids infest the plant. he disease is transmitted to the plant by the aphid vector
Pentalonia nigronervosa and dwarf bananas are very susceptible to this disease. Primary
symptoms of the disease are seen when infected suckers are planted. Such infected suckers
putforth narrow leaves, which are chlorotic and exhibit mosaic symptoms. The affected
leaves are brittle with their margins rolled upwards. Characteristic symptom of bunchy top
virus is the presence of interrupted dark green streaks along the secondary veins of the lamina
or the midrib of the petiole. The diseased plants remain stunted and do not produce bunch of
any commercial value.

The banana aphid (Pentalonia nigronervosa) acquires the virus while feeding on the sap of
infected plants. The virus can persist in the banana aphid for 13-20 days. Banana aphids are
small with reddish-brown, oval-shaped bodies. They have two characteristic projections at
the rear of the abdomen.
The most important factors in controlling banana bunchy top virus are killing the aphid vector
(disease carrier) and rogueing (removing and destroying) infected ba- nana plants

Oregano

Oregano, Origanum vulgare (L.) (Lamiales: Lamiaceae) is an important aromatic plant rich in
terpenoid components used in medical treatments, the food industry , and agriculture . Oregano
essential oil (OEO) has been used for a long time as natural tool to protect against several
microorganisms of stored products , with low animal toxicity and rapid degradation in environment.
Among the antimicrobial properties, OEO exhibits potent insecticidal effects against stored pests . In
T. molitor, deleterious effects caused by synthetic insecticides have been investigated ; however, OEO
might be used to manage T. molitor populations. We assume that OEO causes biocidal effects in
larva, pupa, and adult of T. molitor, which could be due to its capability to affect the survival,
respiration, and behavior of this pest.

Oregano oil is considered as an excellent antiseptic and insect repellent. It has some active ingredients
such as carvacrol, thymol and α-terpinene reported being highly effective in repelling mosquitoes.
Flavonoids (FL) and phenolic acids (PA) are the main types of phenolic compounds present in
oregano ]. Oregano FL and PA have been studied due to their therapeutic potential, which has been
partly attributed to their antioxidant properties. Flavonoids and phenolic acids are molecules
characterized by having at least one aromatic ring with one or more hydroxyl groups attached. FL and
PA are compounds with a wide range of structures and can be classified based on the number and
arrangement of their carbon atoms in: flavonoids (flavonols, flavones, flavan-3-ols, anthocyanidins,
flavanones, isoflavones and others) and non-flavonoids (phenolic acids, hydroxycinnamic acids,
hydroxybenzoic acids, stilbenes and others) and they are commonly found conjugated to sugars and
organic acids . All flavonoids are derived from the aromatic amino acids, phenylalanine and tyrosine
and are C15 compounds arranged in three rings (C6-C3-C6) which are designated as A, B and C .
Their structure varies due to the degree and pattern of hydroxylation, prenylation, alkalization or
glycosylation reactions that modify the primary molecule. These modifications can alter the water
solubility of flavonoids, which can directly affect their bioavailability . Flavones, which are the most
abundant flavonoids present in oregano species, are characterized by the presence of a ketone group
between C-2 and C-3 and the attachment of the B ring to C-2. Among the most widely distributed
flavones in nature are apigenin, luteolin and their derivatives. For further information regarding the
physiological function and health effects of flavones we suggest the studies by Jiang et al. and Singh
et al. On the other hand, hydroxycinnamic acids are part of the non-flavonoid phenolics and the main
subgroup of phenolic acids distributed in the different oregano species . They are formed with an
aromatic ring and a three-carbon chain (C6-C3) . There are four basic structures: coumaric acid,
caffeic acid, ferulic acid and sinapic acid. In nature, they are usually associated with other compounds
such as chlorogenic acid, which is the link between caffeic acid and quinic acid

Oregano oil
Oregano essential oil gives an activity or reaction opposing the pests and insects in plants.
The researchers also found out that lessening the bad odor and making the liquid clear must
be done on the pesticide. In terms of safety, the oregano organic pesticide is safer than the
chemically-based pesticide. Oxidative stress is the disturbance in the pro-oxidant-antioxidant
balance in favor of the former, leading to potential damage; and is often caused by the attack
of reactive species, such as hydrogen peroxide (H2O2), superoxide anion (O2•−), hydroxyl
(HO•), peroxyl (RO2•), and alkoxyl (RO•) radicals, upon the constituents of living organisms .
Oxidative stress can cause an increasing in cell proliferation, adaptation by up-regulation of
defense systems, DNA damage, senescence and cell death. Consequently, many studies have
positively correlated oxidative stress with the pathogenesis of diseases, such as Alzheimer’s
disease, Parkinson’s disease, chronic-inflammation, arthritis, some types of cancer, diabetes
and atherosclerosis, among others. Several phytochemicals have been studied for their
potential scavenging activity towards reactive species and capacity to neutralize oxidative
stress. EOs are among the targeted phytochemicals due to their AOX capacity since it is of
great importance due to their potential to prevent oxidative damage . The AOX properties of
EOs from different species of oregano have been studied; and it is worth to mention that
some of the EOs (carvacrol, thymol) that have been studied in other plant species for their
AOX properties can also be found in different oregano species. Nonetheless, this does not
guarantee their capacity to neutralize oxidative stress and to prevent oxidative damage, since
the differences in the composition of EOO can modify the biological activity .

Objectives
GENERAL OBJECTIVES
This study will explore and assess the effectiveness of Oregano extract as a substitute pesticide
against Pentalonia nigronervosa, an aphid that causes Banana bunchy top virus, to lessen the use of
toxic substances and replaces those with organic and sustainable pesticides.

SPECIFIC OBJECTIVES

1. Determine the efficiency of treatments in terms of preventing growth of Banana bunchy top virus;

2. Determine the level of prevention of different concentrated treatments from Banana bunchy top
virus;

3. Determine the difference among different concentrated treatments in terms of growth of Banana
bunchy top virus;

4. Examine the reaction of bananas on extracts as a treatment against Banana bunchy top virus; and

5. Incorporate the use of organic substances and pesticides in a daily use.

CHAPTER II

Materials and Methods

In this chapter there will be three phases present- Pre-experimentation,
Experimentation and Post-Experimentation.

● PRE EXPERIMENTATION
Preparation
The Proponent must following the laboratory rules and guidelines. Including basic proper
attire inside the laboratory.
Banana Aphids and BBTV Samples
Both the BBTV and Banana Aphids (Pentalonia nigronervosa) will be collected from
TADECO Tagum agricultural development company, Inc. a known company that exports
cavendish bananas internationally. The samples will be tested and confirmed in the
laboratory.
ELISA (Enzyme-linked immunosorbent assay) for diagnosis of banana bunchy top
disease
This process will confirm if the said infected specimen is true.
1. Select the 3rd (third) leaf from the top of the plant, not counting the youngest unfurled
"cigar leaf."

2. Cut a section from the center of the leaf

● EXPERIMENTATION
Extraction of Oregano (Origanum vulgare)
The oregano plant (Origanum vulgare) will be collected from an urban garden located in
Panabo city, Philippines. The oregano samples will be tested and confirmed in the
Department of Agriculture.

The oregano (Origanum vulgare) oil will be extracted using the rotary evaporation. The
vacuum system that is used to separate the substances will result in lowered boiling point and
will eliminate the need for high-temperature distillation. The rotating evaporating flask
increases the surface area of the material that speeds up the distillation process. The forced
convection keeps the substance mixed and heated. By using this process the phytochemical
properties of the Oregano leaves will be separated from the other substances and will be
mixed with water and be accessed by different concentration level.

Petri dish repellency assay

Using different concentration levels of the essential oil, a total of 6 trials using 6 different
concentration levels, from 0.5, 1.0, 1.5, 2.0, 2.5, 3.0. mg/cm^2. In a plastic petri dish size 8
by 1.5cm, filter paper separated into two pieces will be placed in the petri dish to separate the
treated and the untreated area. The number of banana aphids (Pentalonia nigronervosa) also
will be counted.

Concentrat
ion level

Amount of
extract
applied

No. of
aphid
specimen

Direct application of the extract

There are two ways to treat the BBTV, one is killing the Aphid vector (disease carrier) and the other
is roguing (removing and destroying) infected banana plants. In this process the research proponent
applies the treatment directly to the infected leaf. First is disinfecting the whole leaf and apply the
extracted substance into the leaf.

DATA ANALYSIS
Conducting the study and completing all three phases, the proponent will proceed in
reviewing the objectives and hypothesis that was present before the experimentation. The
proponent will start with its conclusion about the study.

● POST-EXPERIMENTATION
The Proponent will complete the remaining laboratory guidelines and rules. And will return the
laboratory to where it was before the proponent conducted its study.
References
Banana Bunchy top virus

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