USING RYEGRASS’ SOIL WITH LEGUME SPECIES AND WHITE SORGHUM

SEEDS ON MAKING ARBUSCULAR MYCORRHIZAL FUNGAL (AMF)

INOCULANT

A Research Paper Presented to

The Facqulty of Science Technology Engineering and Mathematics (STEM) of

Sindangan, National High School in Sindangan, Zamboanga del Norte

In partial fulfillment of the subject Capstone Project

Khryss Franzine A. Fernandez

Zethro Josh A. Carangan
Liezel E. Payag
Althea Faye M. Telebangco
Alexis Ann A. Lomongo
Hilary R. Arboiz

April 2022
 Chapter 1

The Problem and its Scope

Introduction

In today’s time, there has been a problem in the environment especially with plants

where they cannot totally enrich the nutrients uptake coming from the soil. There are factors

that have aggravated the effects of abiotic stresses on plants productivity. These factors are

climate change and agricultural malpractices like the excessive use of fertilizers and harmful

pesticides to the plants.

There will be a big societal impact to people, especially to farmers, that they will

notice that there is no change happening to the growth of a certain plant although they planted

it for a long time. According to Cely et. al (2016), nutrient availability is an important factor

in crop production, and regular addition of chemical fertilizers is the most common practice

to improve yield in agrosystems for intensive crop production. It is widely known that

previous crops affect its growth and yield development through several mechanisms such as

changes in water use efficiency, nutrient use efficiency, soil quality and fertilizer and

chemicals application and many more. However, some aspects cannot be fully explained by

these mechanisms. There are plants that cannot fully attain the increase of nutrients uptake

due to different problems that the plants encounter in their growing process.

In that case, the researchers are solving and supplying solutions to the needs of people

by planning to make an AMF inoculant powder which can help to ensure the growth of water

spinach. It is really a helpful technique to use in order to help the plants survive from
different environmental stresses. For the plants to absorb efficient nutrients in another

opportunity to take advantage of the benefits of mycorrhizae, producing mycorrhizal fungi

inoculant powder is really a good thing for all the people, especially for the farmers. It will

colonize seedlings and increase the impact of yields.

According to Douds et. al, 2008, in less severe circumstances, inoculum may be used

to produce pre-colonized seedlings that can take advantage of mycorrhizae’s benefits from

their first day in the field. Past researches show that this competitive advantage can have a

positive impact on yields. We all know that there are inoculums which are commercials that

are highly expensive. According to Bio-Fit (2017), there has been a remarkable surge in

development of the mycorrhizal-based inoculants market in the last two decades, essentially

in horticulture and field crop production.

The biofertilizer market in agriculture is estimated to reach USD 2.3 billion by 2022,

at a Compound Annual Growth Rate (CAGR) of 14.08% during this period. According to the

study of Fernandez et. al (2017), AMF are especially used in most bioinoculant production as

they have been known for establishing symbiotic relationships with more than 85% of plant

species of agricultural interest. They have been linked to several benefits including macro and

micro-nutrient uptake, water absorption, soil aggregate stability (Ortas et al., 2017) salinity

and drought stress suppression, trace metal detoxification, and protection against pathogens

and herbivores (Smith et al., 2008).

The use of mycorrhizal fungal inoculants is an interesting solution to help plants

withstand difficult environmental conditions. In Cely et. al (2016), it is stated that the use of

some groups of microorganisms that have specific activity providing nutrients to plants is a

good alternative, and AMF enhances plant nutrition by providing especially phosphorus

improving plant growth and increasing crop production. They are very useful to help plants
survive and grow despite the excessive heat and lack of water they may experience more

often due to climate change, especially in urban environments.

This biofertilizer inoculant will benefit significantly to people especially farmers,

gardeners, producers of vegetables and fruits, and many more. That's why the purpose of this

study will be producing inexpensive inoculum out of effective species such as white sorghum

seeds, patani seeds, and dun pea seeds with ryegrass plant soil which will be varied in

different numbers of seeds and constant amount of soil.

Framework of the Study

This study is supported by Bagyaraj and Ashwin (2017), in their work, reported

significant increases in crop yield following inoculation with AMF which aid in stimulating

key effects such as root development, improved soil structure, increased nutrient uptake, and

mobility of ions.

Besides enhancing plant tolerance to stresses, endomycorrhiza improves the general

well-being of plants. They occur in most ecosystems of the world and are found in many

important crop species such as wheat, maize, rice, grape, soybean, and cotton and in

horticultural species like roses and petunias. Commonest among them is AMF.

Arbuscular mycorrhizal fungi (AMF) play a key role in plant growth promotion, plant

protection, and soil quality improvements. According to the study carried out by Koller et al.

(2013), AMF colonizes the cortical cells of vascular plants where arbuscular structures are

developed, which allow for the exchange of nutrients among the symbionts. Moreover, are

resistant against soil-borne pathogens, interest in the use of these fungal agents as

biofertilizers has been on the increase (Odo 2017; Sumita et al., 2015; Aggani, 2013).

Odoh et.al (2020) detailed that among the fungal biofertilizers such as Fusarium

species pluralis, Trichoderma species pluralis, etc, mycorrhizal biofertilizers products greatly
appeal to the agro-industry mainly due to its versatility and use of environment friendly

technology.

Legumes are valuable crops for food, fodder, green manure and biofuel. Although

some legumes may store large mass fractions of starch in their seeds, legumes are generally

prized for their protein rich biomass and seed protein and lipid mass fractions. Particularly,

this study will use patani and dun pea seeds.

Grasses are monocotyledonous plants in the family Poaceae also known as

Gramineae. Some species are grown as agricultural crops, and these are among the most

important foods for humans and domestic livestock. Particularly, this study will use white

sorghum seeds.

Furthermore, Change Your World (2017) showed that over 160 commercially

important plant groups that benefit from endomycorrhizal fungi which includes all beans,

peas, wheat, sorghum, etc. Additionally, The Sunseed Dessert Technology specified that a

good combination on multiplying the mycorrhiza would be a grassy species (eg maize, millet,

sorghum, oats, wheat) or an allium (onion, leek), with a species of legume (beans, peas,

lentils, alfalfa, clover).

Figure 1 shows the Conceptual Paradigm of the Study. On the left side, it represents

the independent variable which displays the different number of legume species seeds (8,6,4)

and white sorghum seeds (8,6,4,) with the following seed combinations: patani seeds and

white sorghum seeds; dun pea seeds and white sorghum seeds. The left side also represents

the control group which shows the constant amount of soil (500g) and water (500mL). Thus,

the right side represents the dependent variable which shows the water spinach’s growth per

week after being inoculated in terms of its height and number of leaves. It also displays the

water spinach’s growth per week without inoculation in terms of its height and number of

leaves.
 Independent Variable

Different amount of legume species seeds

(8,6,4) and white sorghum seeds (8,6,4,)
with the following seed combinations:

Dependent Variable
A. Patani and white sorghum
B. Dun pea and white sorghum
The growth of the water
spinach plant per week after
being inoculated in terms of:

a) Height
b) Number of leaves

The water spinach’s growth

per week without
Control Group
inoculation in terms of:

a) Height
b) Number of leaves

Amount of soil (500g),

Amount of water (500mL)

 Figure 1. Conceptual Paradigm of the Study
Statement of the Problem

This study will be conducted to determine if ryegrass’ soil with legume species seeds

such as patani and dun pea seeds, and white sorghum seeds can be used as an alternative

source of making AMF inoculant. It specifically aims to know the following:

1. What is the mean of the different amount of seeds in terms of:

a. Patani and white sorghum seeds

b. Dun pea and white sorghum seeds?

2. What is the mean of the growth of the water spinach plant per week after being

inoculated in terms of:

a. height

b. number of leaves?

3. What is the mean of the growth of the water spinach plant per week without being

inoculated in terms of:

a. height

b. number of leaves?

Null Hypothesis

Ho1: There is no significant difference between the different amount of seeds in terms

of:
 a. White Sorghum and Patani Seeds

b. White Sorghum and Dun Pea Seeds

Ho2: There is no significant difference between the growth of the water spinach plant

per week after being inoculated in terms of:

a. height

b. number of leaves

Ho3: There is no significant difference between the growth of the water spinach plant

per week without being inoculated in terms of:

a. height

b. number of leaves

Scope and Delimitation of the Study

The study will be conducted to know if ryegrass’ soil with legume species and white

sorghum seeds that can be an alternative source of making an AMF inoculant in terms of the

different amount of seeds and constant amount of soil.

The soil needed in the study will be collected in a specific area at Sindangan National

High School, Dapaon, Sindangan Zambaonga del Norte. The study will be conducted at

Disud, Sindangan, Zamboanga del Norte for materials and equipment needed for the study

are already available there. Furthermore, the location makes it easier for the researchers to

plant and monitor the products.

Significance of the Study

The study aims to make an alternative source of making AMF inoculant out of

ryegrass’ soil with different grass and legume species seeds such as white sorghum seeds,
patani seeds, and dun pea seeds and test its effectivity. It could offer a significant help to the

following groups of people:

The farmers. All the farmers are always struggling because of the environmental

issues that they are always facing that can affect the growth of their plants. With this

alternative AMF inoculant, it can help them to ensure the growth of their certain plant and

help the plants to survive from different environmental stresses.

Gardeners. Some gardeners are having a problem of the unhealthy growth of their

plant because of the chemicals and harmful pesticides that affect its health. With the use of

this alternative AMF inoculant, it will help to totally enrich the nutrients uptake coming from

the soil that can help the plant to grow healthier.

Agricultural manufacturers. This study could introduce a new source of making

inoculant for easy and cost-efficient production.

Definitions of Terms

To understand and clarify the terms used in the study, the following are hereby

defined. The variables involved form theoretical and operational definitions of the concepts

to be investigated in the research.

Dun Pea. Pisum sativum, sometimes known as pea, is an annual herbaceous legume in they

Fabaceae family that is farmed for its edible seeds and seed pods. Pea plants can be

bushy or climbing, with slender stems that use tendrils to connect to a substrate. Each

leaf contains one to three pairs of oval leaflets and can grow to be 1–6 cm long.

White, red, or purple blooms bloom on the shrub, which has bloated or compressed

green seedpods that can be straight or curved. The pods can be anywhere between 4

and 15 centimeters long and 1.5–2.5 centimeters broad. Each pod contains 2 to 10

seeds, sometimes known as peas. The pea plant is an annual that only lasts one
 growing season and grows to a height of 30–150 cm. Garden pea is another name for

pea (CABI Crop Protection Compendium, 2014). In this study, it serves as one of the

legume species seeds in making AMF inoculant.

Patani. Patani is a climbing, slender, annual, smooth, sparingly hairy, herbaceous vine

reaching a length of 4 or more meters. Leaves are thin, compound with three leaflets

which are ovate, 6 to 12 centimeters long, rounded at the base and pointed at the tip.

Studies have suggested hypolipidemic, hypoglycemic, antibacterial, antifungal,

antiproliferative, estrogenic, hepatoprotective, nephroprotective, antihypertensive,

herbicidal, antioxidant properties (Godofredo, 2022). In this study, it serves as one of

the legume species seeds in making AMF inoculant.

Ryegrass’ soil. A vigorous cool-season grass with an extensive root system. As a cover crop,

annual ryegrass helps prevent erosion, builds soil organic matter, improves soil tilth,

captures residual nitrogen and can significantly increase the rooting depth of corn and

soybeans. This guide covers the management practices essential to growing a

successful annual ryegrass cover crop – timetested strategies from more than 13 years of

on-farm testing in the Midwest. Using annual ryegrass as a cover crop requires proper

management. It must be seeded in a timely manner, at the proper rates and it must be

controlled on time so that it does not compete with corn or soybeans (N.A., 2016).

In this study, it is used as the starter soil in mixing it with the legume species seeds

and white sorghum seeds.

White sorghum. a cereal grain that grows tall like corn and is utilized for a variety of

purposes other than sweetening. Sorghum is first and principally utilized as animal

feed and then converted into ethanol in the United States. Because it is drought

tolerant, it is a popular crop to raise in the drier parts of the United States. It has also
been a prominent crop in Africa, where it has been grown for over 4,000 years.

Actually, sorghum is supposed to have arrived in America via slave ships from Africa

(Matt, 2022). In this study, it serves as grassy specie in making AMF inoculant.
 Chapter 2

Review of Related Literature

This chapter presents the synthesis of the related literature and related studies about

the study. The variables used in the study are separated in their uses in each study.

Inoculation

The first interaction of a pathogen with a plant location where the infection is feasible

is known as inoculation. The inoculum is the pathogen(s) that lands on or else come into

contact with the plant. Any component of the pathogen that can cause infection is called an

inoculum. As a result, spores, sclerotia (i.e., a compact mass of mycelium), or fragments of

mycelium may be used as inoculum in fungi. The inoculum is always entire individuals of

bacteria, mollicutes, protozoa, viruses, and viroids, respectively, in bacteria, mollicutes,

protozoa, viruses, and viroids. The inoculum in nematodes might be adult nematodes,

nematode juveniles, or eggs. Plant pieces or seeds may be used as inoculum in parasitic

higher plants. The inoculum can be made up of a single pathogen individual, such as a spore

or a multicellular sclerotium, or millions of pathogen individuals, such as bacteria carried in a

drop of water. A propagule is a single unit of inoculum from any pathogen.

There are types of inoculum, a primary inoculum is an inoculum that remains dormant

over the winter or summer and causes the original diseases in the spring or autumn. The

infections it causes are known as primary infections. A secondary inoculum is an inoculum

that is created as a result of primary infections and produces secondary infections. The more

prevalent the primary inoculum is and the closer it is to the crop, the more severe the illness

and the resulting losses are.

In several fungal and bacterial infections, the inoculum is formed on the branches,

trunks, or roots of perennial plants, such as shrubs and trees. The inoculum can be found in

the plant waste or soil of the crop's growing field; it can also be brought into the field with

seed, transplants, tubers, or other propagative organs, or it can arrive from outside the field.

Inoculum can come from neighboring plants or fields and fields thousands of kilometers

distant. The inoculum lives in perennial weeds or alternate hosts in many plant diseases,

especially those that affect annual crops, and it is transmitted from them to annual and other

plants every season. Fungi, bacteria, parasitic higher plants, and nematodes either create their

inoculum on the surface of diseased plants, or when the infected tissue breaks down, their

inoculum reaches the plant surface. Viruses, viroids, mollicutes, fastidious bacteria, and

protozoa develop their inoculum within plants; in nature, this inoculum virtually never

reaches the plant surface, therefore it can only be transported from one plant to another by a

vector, such as an insect (Agrios, 2005).

When a high number of live bacteria must be introduced, soil inoculation is

beneficial. Many restrictions associated with other related procedures are removed by using a

high load of microorganisms in soil inoculation. At the time of sowing, granular inoculants

with a diameter of 0.5 to 1.5 mm are retained in the seedbed with the seed (Mahanty et al.,

2016). Soil inoculation is a more expensive but more practical technique of delivering

bioformulation in the field. Mixing granular, powdered, or encapsulated bioformulation with

soil is one way to do so (Bashan, 1998).

Arbuscular Mycorrhizal Fungal

 Arbuscular Mycorrhizal Fungal (AMF) is soil-borne fungi that can boost plant

nutrient uptake and resilience to various abiotic stresses (Sun et al., 2018). The sub-phylum

Glomeromycotina of the phylum Mucoromycota is home to the bulk of AMF species

(Spatafora et al., 2016). This sub-phylum contains four orders of AMF, namely Glomerales,

Archaeosporales, Paraglomerales, and Diversisporales, as well as 25 taxa (Redecker et al.,

2013). They are obligate biotrophs that eat plant photosynthetic products and lipids to

complete their life cycle (Bago et al., 2000). (Jiang et al., 2017). AMF-mediated growth

promotion protects plants from fungal infections as well as enhances water and mineral

nutrient intake from the surrounding soil (Smith and Read, 2008; Jung et al., 2012). Bio-

fertilizers are a blend of naturally occurring compounds used to boost soil fertility. These

nutrients are beneficial to both soil health and plant growth and development (Sadhana,

2014). AMF has been the subject of numerous research studies over the last two decades, all

of which have demonstrated its numerous advantages to soil health and crop productivity.

Because mycorrhizal treatment may significantly reduce the quantitative usage of chemical

fertilizer input, especially phosphorus, it is widely assumed that AMF could be considered as

an alternative for inorganic fertilizers shortly (Ortas, 2012).

Arbuscular mycorrhizal fungi (AMF) help host plants grow more vigorously under

stress by mediating a series of complex communication events between the plant and the

fungus, resulting in increased photosynthetic rate and other gas exchange-related traits, as

well as increased water uptake (Birhane et al., 2012). Several studies have found that fungal

symbiosis improves resilience to several stressors, including drought, salt, herbivory,

temperature, metals, and pathogens (Rodriguez et al., 2008; Ahanger et al., 2014; Salam et

al., 2017). Vesicles, arbuscules, and hyphae generate vesicles, arbuscules, and hyphae in

roots, as well as spores and hyphae in the rhizosphere.

 Continuous application of inorganic fertilizers, herbicides, and fungicides has resulted

in a slew of issues for soil, plants, and human health, owing to their negative influence on

food quality, soil health, and air and water systems (Yang et al., 2004). AMF is thought to be

capable of reducing chemical fertilizer consumption by up to 50% for optimal agricultural

productivity, however, this estimate is dependent on the type of plant species and the

prevailing stressful regimes.

AMF's helpful impact in promoting plant growth in stressed situations has already

been shown in a few research publications. As a result, the current material on the role of

AMF has been combined cohesively in this review to gain a better understanding of AMF's

symbiotic interaction with a range of plants under stressful situations. Plants inoculated with

AMF may effectively combat many environmental cues, such as salinity, drought, nutrient

stress, alkali stress, cold stress, and extreme temperatures, and so help increase per hectare

yield of a wide variety of crops and vegetables.

Encouragement of the use of AMFs is critical for the long-term viability of modern

global agricultural systems. Without a doubt, using AMF for agricultural enhancement can

help to reduce the use of synthetic fertilizers and other chemicals, encouraging bio-healthy

agriculture. AMF-mediated growth and productivity enhancement in crop plants may be

useful in meeting the global population's growing consumption demands. Furthermore,

because of their widespread use, environmentally friendly technologies will be strongly

promoted. The identification of genes and gene products mediating AMF-mediated growth

and development regulation under stressful stimuli should be the primary focus of future

studies.

Ryegrass
 Ryegrass (Lolium) is a grass genus with roughly ten species in the Poaceae family. In

temperate Eurasia and Africa, a variety of species are planted as forage and lawn grasses, and

perennial ryegrass (Lolium perenne) and annual ryegrass (Lolium multiflorum) are key

components of pasture and lawn-seed mixtures used around the world. The plants have no

relation to rye cereal (Secale cereale). Ryegrasses come in two varieties: annuals and

perennials. Tough dark green leaves cover the tufted plants, which grow to be around 0.3 to 1

meter (1 to 3.3 feet) tall. The flower spikelets form a zigzag rachis as they expand (flower

stem). The plants have deep root systems that help with erosion control (The Editors of

Encyclopedia Brittanica, 2019).

Perennial ryegrass is native to central Asia, the Middle East, North Africa, and

southern Europe, from Bulgaria in the east to France in the west. Perennial ryegrass is a cool-

season perennial bunchgrass with a C-3 metabolism that grows best in moderate climates. It

produces just tillers and has a limited capacity to spread as a bunchgrass. The number of

diploid chromosomes in turf-type perennial ryegrass is 14. It has an annual root system,

which means that during the spring flush growth phase, the bulk of its roots is replaced year

(Tom Cook of College of Agricultural Sciences, 2022).

Annual ryegrass, in southern Australia's annual winter cropping systems, it's one of

the most serious and costly weeds. Annual ryegrass is a tough competitor, and it may fight

with crops as early as the two-leaf stage. It's a weed that grows from late autumn to early

spring. Initial seed bank levels, as well as the frequency and amount of rainfall, affect the

number of emergence flushes and the density of plants that emerge (Peltzer, 2021).

Legume

Legume (Fabaceae), also known as a pod, is the fruit of pea plants (Fabaceae).

Although some legumes, such as peanuts (Arachis hypogaea) and carobs (Ceratonia siliqua),
do not naturally open, most are dehiscent fruits that release their seeds by splitting open along

two seams. The fruits vary in a range of sizes and shapes, but many are long and narrow with

a single line of seeds. The monkey ladder (Entada gigas) carries the largest legumes, which

can grow up to 2 meters (6.6 feet) in length. The legumes of certain food crops, such as snow

peas (Pisum sativum variety), edamame (Glycine max), and green beans (Phaseolus vulgaris),

are harvested when they are dry, papery, or hard and woody at maturity (The Editors of

Encyclopedia Brittanica, 2021).

White Sorghum

Sorghum (Sorghum bicolor), often known as big millet, Indian millet, milo, durra, or

orshallu, is a grass family (Poaceae) cereal grain plant with edible starchy seeds. The plant is

thought to have originated in Africa, where it is an important food crop. It comes in a variety

of forms, including grain sorghums for food, grass sorghums for hay and fodder, and

broomcorn for brooms and brushes. Sorghum is known as jowar, cholam, or jonna in India,

Guinea corn in West Africa, and kaoliang in China. Sorghum is prized for its drought and

heat endurance in hot and arid climates. Sorghum is tough grass that grows to a height of 0.6

to 2.4 meters (2 to 8 feet), with some plants reaching 4.6 meters (15 feet). White wax coats

the stalks and leaves, and the pith, or inner portion, of some types' stalks, is juicy and sweet.

The leaves are 76 cm (2.5 feet) long and 5 cm (2 inches) wide. Each flower cluster yields

800–3,000 kernels and is formed in panicles that range from loose to dense. The seeds vary in

color, shape, and size depending on the variety, but they are smaller than wheat seeds (The

Editors of Encyclopedia Brittanica, 2019).

Patani

Patani (Phaseolus lunatus) or known as lima bean, is the edible seeds of the lima bean

(Phaseolus lunatus L.) are grown in tropical and subtropical climates. Phaseolus lunatus
comes in both wild and cultivated varieties, which are known as Phaseolus lunatus var.

Phaseolus lunatus var. Silvester Baudet and Phaseolus lunatus var. Lunatus, to be precise.

Lima bean is a herbaceous plant that grows in two different ways. The perennial form is an

indeterminate, robust climbing and trailing plant with only axillary flowers that grow up to 2-

6 m tall. It has 2 m long swollen and meaty roots. The annual lima bean is a bushy, pseudo-

determinate plant that grows to be 0.3-0.9 meters tall with terminal and axillary flowering. Its

roots are very thin, lima bean sprouts, leaves, young pods, and green seeds (immature or dry)

are all edible and can be consumed as a vegetable. The dry seeds are used in soups and stews

and are cooked, fried, pounded into powder, and baked. After the harvest, the vines, leaves,

and empty pods can be used as fodder and turned into hay or silage. Lima beans can be

utilized as a cover crop or as green manure. Although only a few cultivars are appropriate for

intercropping, the lima bean could be useful in intercropping systems (Baudoin, 2006).

Dun Pea

Dun Pea is classified as a bean because it belongs to the Fabaceae family. Dun pea is

a pea cultivar that belongs to the field pea family (Pisum sativum). Field peas are one of the

oldest domesticated crops, having been farmed in Turkey for at least 7.000 years. Dun peas

are currently widely cultivated, with almost 25 million acres planted worldwide. Russia and

China are the leading producers of a dun pea, followed by Canada, Australia, the United

States, and Europe. Dun peas get their name from the color of the pea, which is brown (dun).

Dun peas have dimpled forms and are greenish-brown in color. People frequently confuse it

for cowpea because of its description. Because this pea is a cool-season legume crop, many

farmers in warmer climates produce it. Because this pea is a cool-season legume crop, many

farmers in warmer areas plant it under regulated conditions. Field peas are commonly

consumed by humans or used as livestock feed. Dun peas are a legume with high nutritional
value, containing Vitamin B, C, and K, as well as folic acid and fiber. This pea has a pleasant

flavor that makes it suitable for human eating (Tradeasia International, 2021).

Chapter 3

Methodology

Research Design

The researchers will conduct this study to have an effective AMF inoculant to

improve the growth of water spinach (kangkong). This study will be using experimental

design; specifically, the two-group design involving a treatment with 2 or more levels.

Moreover, the proponents will use this type of experimental design because control group and

experimental group are involved in this study.

Research Locale

The soil needed in the study will be collected in a specific area at Sindangan National

High School, Dapaon, Sindangan Zambaonga del Norte. Moreover, this study will be

conducted in the residence of Liezel Payag, located in Disud, Sindangan Zamboanga del

Norte, because the materials are available there. Also, the location makes it easier for the

researchers to plant and monitor the products.

Research subject

This study is all about making AMF inoculant made of ryegrass soil with different

legume species seeds such as patani and dun pea seeds, and white sorghum seeds. The

researchers chose these following variables as their subject in the study since
endomycorrhizal or arbuscular mycorrhizae form with most grassy species and legume seeds.

Besides that, these certain types of species can immediately grow on soil. Also, AMF are

commonly known as biofertilizers. Moreover, it is widely believed that the inoculation of

AMF provides tolerance to host plants against stressful situations like heat, salinity, drought,

metals and extreme temperatures (Begum, 2019).

The researchers come up to this idea on making an AMF inoculant which is

inexpensive to the people especially the farmers on helping their plants grow healthy and

increase the impact of yields.

Experimental Procedure

The researchers will get all the materials needed in conducting the experiment. First,

they will be going to perform the following steps in producing arbuscular mycorrhizal fungi

inoculant:

a. Soaking of white sorghum, patani and dun pea seeds

The researchers will soak the seeds for 12 hours and will prepare the same sized bowl

where they will put all the seeds that need to be soaked. The three bowls will have the same

amount of seeds that will be poured and the same amount of water. Soaking seeds before

planting helps to break down the seeds’ natural defenses against what it expects from the

environment, which then allows the seeds to germinate faster and speed up seed

establishment with a corresponding increase in survival rates when different stresses will

make contact with them while growing. After the 12 hours of soaking the seeds, the

researchers will get all the seeds which are all ready to be planted.

b. Collecting the starter soil

 First, they will prepare all the necessary equipment in collecting the starter soil such

as weighing scale, shovel, sacks, and gloves. Second, the researchers will find a good

condition of ryegrass plant soil which did not undergo inoculation. Third, they will clear

away 35cm by 35cm of the vegetation underneath the ryegrass plant. After that, they will dig

down using a shovel to a depth of about 15cm collecting the ryegrass soil and as many fine

roots as possible. Moreover, will get all the stones from the collected soil. After getting the

stones on it, they will weigh approximately 5kg of the ryegrass soil and put it on the prepared

sacks.

c. Setting the pots

The method that the researchers will use in multiplying the collected soil with

mycorrhizae is using the same sized pots. First, they will prepare the starter soil, black plastic

pots, and other equipment. Second, they will separate the pots and put it on each trial in every

set-up of pairs. Third, the researchers will put the collected soil on it and make sure that the

pots with soil will be ready to be planted. They will be having 3 trials for each pair of grass

and legume species seeds namely: patani and white sorghum seeds and dun pea and white

sorghum seeds which will differ in number of seeds and constant amount of soil.

d. Planting the seeds

After making the set-up of the pots, they will be preparing for the exciting part of the

study, the planting of the seeds as bait plants. First, they will prepare the required amount of

seeds that have already been soaked in water. Second, the researchers will plant the two pairs

of seeds namely: white sorghum and patani seeds, and white sorghum and dun pea seeds.

They will be going to sew it closer than normal and in a random method of sowing them.

They will water the plants regularly with a constant of water (500mL).

e. Collecting the inoculant

 After one month of planting the seeds as bait plants, the researchers will now collect

the AMF inoculant. They will cut down the base of the bait plant’s stem and stop watering it.

This will kill the plant and trick the fungus into producing reproductive spores. Then, they

will wait for ten days for the inoculant to be prepared by pulling up the roots of the bait

plants. They will chop into roughly 0.5 cm and then will mix back into the soil from the pots.

This will be now the produced arbuscular mycorrhizal fungi inoculant. After that, they will

crush it using mortar and pestle to attain the required type of inoculant which is the powder

one.

Second, the researchers will apply the produced AMF inoculant to their chosen target

plant which is water spinach or kangkong. They will prepare the 3 kangkong seeds in each

trial of the pair. Then, they will set-up the same planting method, which is the use of pots, but

they will not use the present of ryegrass soil this time. They will have to have a type of soil

where there will be no mycorrhizae present on that soil and it did not undergo inoculation.

The reason for this is to know and see the effectiveness of their AMF inoculant that they

produced. They will compare the presence of AMF inoculant on kangkong seeds to without

the application of produced inoculant. Before sowing the seeds, they will apply the AMF

inoculant just a half part to each seed. They will not cover the whole seed with inoculant and

will sow these seeds into the pots with the chosen type of soil closer than normal and in a

random method. They will water it regularly as what they did in planting the bait plants.

Data Gathering

Different grass and legume species seeds such as white sorghum, patani and dun pea

seeds with ryegrass soil will be used in gathering the data of the study. These seeds will be

used in the study because of its ability to penetrate mycorrhizae inside and outside the

cortical cells. Besides that, they are also fast-growing plants which have the presence of
different nutrients such as magnesium, calcium, phosphorus, and many more. The ryegrass

plant soil with mycorrhizae will be used as where the plant’s seeds will be planted. These will

be the components that the researchers will need in order to stabilize and achieve the most

effective combination of pairs between grass and legume species seeds namely: white

sorghum and patani seeds and white sorghum and dun pea seeds. The making of AMF

inoculant will be varied in different numbers of seeds and constant amount of ryegrass soil.

The data will be gathered by the researchers to determine which pair is the best

combination of inoculant to kangkong seeds in terms of the height of the plant and the

number of leaves. After preparing all the set-ups, the researchers will be going to test the

AMF inoculant’s effectiveness to kangkong seeds which will be compared to the same seeds

without the application of the produced inoculant in terms of the following:

a. height of the plant for each set-up (with and without AMF inoculant)

In each trial of pairs, they will have 3 kangkong seeds to use. The researchers will test

the height by measuring it through a tape measure one by one. They will measure it per week

after the plants are being inoculated. Moreover, they will calculate its mean or average height

for it to know the exact measurement on that day. They will have a sheet of paper to use

where all the measurements will be indicated.

b. Number of leaves

The researchers will know the plant’s number of leaves by means of counting its

leaves per week. They will not consider counting a leaf if it will not reach to 1cm of the

measurement. They will calculate the average number of leaves in each trial of pairs and

write it down on the provided sheet.

Treatment of Data
 This study is all about producing an arbuscular fungal inoculant with the use of

different species of grass and legume seeds. The researchers will use the ANOVA or

Analysis of Variance test specifically the one-way ANOVA. This study contains more than 2

populations wherein there is one independent variable namely : Different amount of legume

species seeds(8,6,4)and white sorghum seeds (8,6,4)with the following seed combination:

patani and white sorghum: and dun pea and white sorghum, two dependent variable such as

the growth of the water spinach plant per week after being inoculated (height, number of

leaves and the spinach’s growth per week without inoculation in terms of: height, number of

leaves and thickness of stem and lastly the control group: the amount of soil(500g) and the

amount of water(500mL).