PRODUCTION AND EVALUATION OF BIOFERTILIZER FOR SUSTAINABLE AND GREEN AGRICULTURAL PRACTICES

OF BLACK PEPPER

Nabila Binti Satibi

Master of Environmental ScienceS (Land Use and Water Resource Management)654.5

2014N1I6 2014

Pusat Khidmat Maklumat Akademik UNIVERSITI MALAYSJA SARAWAI<

Production and Evaluation of Biofertilizer for Sustainable and Green Agricultural Practices of Black Pepper

I

NABILA BINTI SATIBI

A dissertation submitted in partial fulfilment of the requirements for the degree of Master in Environmental Science

(Land Use and Water Resource Management) I

Faculty of Resource Science and Technology

UNIVERSITI MALAYSlA SARA W AK

2014

ACKNOWLEDGEMENT

, First of aU, I would like to thanks to Allah S.W.T for giving me patience and

strength to complete this study.

I would like to convey my deepest gratitude and thanks to my parents, Mr. Satibi

bin Arabi and Mdm Samsiah binti Hj. Nawawi who always helping and supporting me

both in financial and morale. Also thanks to my friends, especially Eza Azuren for

passionately supporting and encouraging me during this research study. They have given

me full encouragement from the beginning until the completion of this study_

Also, I would like convey my warmest appreciation to my supervisor, Assoc. Prof

Dr Awang Ahmad Sallehin Awang Husaini who had been supervising and teaching me

throughout my research study period. I would like to thanks Universiti Malaysia Sarawak

(UNlMAS) for the facilities provided.

Finally, to all people that were involved directly and indirectly. I would not able to

complete my research studies without support and guidance from all of them.

DECLARA TION

I hereby declare that no portion of this dissertation has been submitted in support of an

application for another master of qualification of this or any other university or institution

ofhigher learning.

i ..

a·· (Nabila Binti Satibi) SLUSE-M Programme Faculty ofResource Science and Technology

Universiti Malaysia Sarawak

Date: . .'.~:.C:>~:. ?-<?ti ...... .. .. .

P USit Kliidmat Maklumat Akadrmik UNIVERSm MALAYSIA SAKAWAK

TABLE OF CONTENT

Page

Table of Content 1- 11

List of Abbreviations III

List of Table iii

List of Figure IV

Abstract V

1.0 INTRODUCTION

1.1 Problem Statement 4

1.2 Objectives 5

2.0 LITERATURE REVIEW

2.1 Black Pepper 6

2.1.1 Pepper Cuttings for Propagation 7

2.2 Biological Control 8

2.3 Biofertilizers 10

2.3.1 Previous Study on Biofertilizers in Black Pepper 11

2.4 Phytopthora capsici 12

2.4.1 Phytopthora blight 13

2.5 Previous Study using Bacteria Burkholderia unamae and

Enterobacter cloacae 14

2.5.1 Burkholderia unamae P10 14

2.5.2 Enterobacter cloacae P11 15

2.6 Metabolite 16

2.7 Bacterial Count 17

2.8 Chlorophyll 18

2.8.1 Chlorophyll Analysis 19

3.0 METHODOLOGY

3.1 Study Sites 21

3.1.1 Study Site for Field Test 21

3.1.2 Sample Collection for Greenhouse Trial 21

3.2 Blofertilizers Production 22

3.3 Laboratory Work, Greenhouse Trial and Field Test 23

3.3.1 Laboratory work I

3.3.1.1 In vitro test 23

3.3.1.2 Bacterial count 23

3.3.1.3 Burkholderia unamae and Enterobacter cloacae Metabolite 24

3.3.1.4 Chlorophyll a Analysis 24

3.3.2 Greenhouse Trial

3.3.2.1 In vivo evaluation 25

3.3.3 Field Test

3.3.3.1 In vivo evaluation 25

3.4 Data Analysis 25

4.0 RESULTS & DISCUSSION

4.1 In vitro test 27

4.2 Bacterial Count 27

4.2.1 Burkholderia unamae P10 28

4.2.2 Enterobacter cloacae P11 29

4.3 Greenhouse Trial 30

4.3.1 Length of Leaves 31

4.3.2 Width of Leaves 33

4.3.3 Length of Root and Height of Pepper 35

4.3.3.1 Root Length of Pepper Cuttings 35

4.3.3.2 Height of Pepper Cuttings 38

4.4 Field Test 40

4.4.1 Changes in Morphology 40

4.4.2 Chlorophyll a Analysis 42

5.0 CONCLUSION 45

References 47

Appendices 51

11

List of Abbreviations

" CMS Cahaya Mata Sarawak .I

LB Luria Broth

MEA Malt Extract Agar

PI0 Burkholderia unamae PI0

PH Enterobacter cloacae Ptt

List of Table

Page

Table 1.0 : Characteristics of recommended Pepper Varieties in 2

Sarawak (Adopted from Agriculture Research Centre,

2006) . Table 2.0 : Treatments on pepper cutting 27

Table 3.0 : One-way ANOVA of Length of Leaves 31

Table 4.0 : One-way ANOV A of Width of Leaves 33

Table 5.0 : One-way ANOV A of Length of Root 36

Table 6.0 : One-way ANOV A of Different of Cuttings Height 38

Table 7.0 : Changes in Morphology After t M.onth Application with 40

Treatments

Table 8.0 : Descriptive Table of Chlorophyll a Analysis 42

Table 9.0 : One-way ANOV A of Chlorophyll a Analysis 43

1Il

List of Figure

Page

Figure 1.0 : Metabolite production 16

Figure 2.0 : Absorption spectra of chlorophyll a and b 19

Figure 3.0 : Absorption spectrum of chlorophyll a 20

Figure 4.0 : Weigh ofCMS Compost 22

Figure 5.0 : Bacterial count on Burkholderia unamae P10 28

Figure 6.0 : Bacterial count on Enterobacter cloacae PH 29

Figure 7.0 : Pepper cutting being attacked by snail 30

Figure 8.0 : Mean Plot of Length of Leaves 31

Figure 9.0 : Mean Plot of Width of Leaves 33

Figure 10.0 : Root Lengths of Four Treatment 35

Figure 11.0 : Mean Plot of Length of Root 36

Figure 12.0 : Mean Plot of Different of Cuttings Height 38

Figure 13.0 : Mean Plot of Chlorophyll a Analysis 43

IV

Production and Evaluation of Biofertilizer for Sustainable and Green Agricultural Practices of Black Pepper

Nabila Binli Salibi

SLUSE-M Programme Faculty of Resource Science and Technology

Universiti Malaysia Sarawak 94300 Kota Samarahan, Sarawak

Abstract

~Black pepper is famous as king of spices and it has value due to its better pungency and aroma. This black pepper also known as peppercorn and its scientific name is Piper nigrum Leonian (P. nigrum L.). Various diseases attacked this black pepper but most common disease is root rot caused by Phytophthora capsici. Main objective of the study is to implement green agricultural practices to protect and conserve the environment by using land for agricUltural practices in sustainable wa, Bacteria Burkholderia unamae PIO (Bioactiv-SFl) and Enterobacter cloacae PH (Bioactiv-SF2) were used to formed biofertilizers which have been applied to black pepper. Serian has been selected as study sites and data on morphology such as number of leave, diameter of leave, height of plant and length of roots of black pepper were recorded for greenhouse trial while color of leaves, height of tree, number of leaves, fruit production and health condition of the tree for field test. For conclusion, T 4 show the best result compared with T 3.

Key words: Black pepper, Phytophthora capsici, Burkholderia unamae, Enterobacter cloacae, biofertilizers

Abstrak

Lada hitam terkenal sebagai raja rempah dan ia mempunyai nitai kerana aroma dan kepedasan yang lebih baik. Lada hitam ini juga dikenali sebagai 'peppercorn' dan nama saintiflk bagi laga hi/am adalah Piper nigrum Leonian (p. nigrum L.). Pelbagai jenis penyakit menyerang lada hi/am tetapi penyakit yang biasa menyerang ialah akar repllt yang disehahkan oleh Phytophthora capsici. Objektif utama penyelidikan ini ialah untuk melaksanakan perlanian hijau bagi melindungi dan memelihara persekitaran dengan menggunakan kawasan tanah untuk amalan pertanian yang mampan. Bakteria Burkholderia unamae PIO (Bioactiv-SFl) dan Enterobacter cloacae Pll (Bioactiv-SF2) digunakan untuk menghasitkan bioJerlilizers yang akan diaplikasikan kepada pokok lada hi/am. Serian telah dipilih sebagai kawasan kajian dan maklumat seperli jumlah daun, diameter dalln, tinggi pokok dan panjang akar lada hi/am akan direkod bagi kajian di rumah hijau manakala warna daun, tinggi pokok, jumlah' daun, penghasilan buah dan kondisi kesihatan pokok direkod bagi kajian di ladang lada di Serian. Kesimpllian hagi kajian ini, T4 menunjukkan keputllsan yang paling baik jika dihandingkan dengan h

Kata kunci : Lada hi/am, Phytophthora capsici, Burkholderia unamae, Enterobacter cloacae, biofertilizers

v

1.0 INTRODUCTION

Originally black pepper comes from Kerala, India where it then spread to otller country of

South and Southeast Asia. It becomes an important source of spice for the country. Hindu

colonists that migrate from India brought cuttings of black pepper to Indonesia and other

countries. It is recorded that India has the largest area of pepper plantation approximately

177, 340 ha which in Kerala alone accOlmts 95% ofthe plantation (Sivaraman et al., 1999).

Black pepper not only widely used as spices that are essential seasoning in food but it also

used for traditional medicine. For example, b1ack pepper has been used to treat coughs,

colds, breathing, heart problems, and stomach ailments in Ayurvedic medicine. Other use

ofblack pepper is it can produce pepper oil and oleoresin which both of it are used in food

industry.

Malaysia is one of the countries that cultivate pepper and earning foreign exchange from

pepper industry. Malaysia is one of the largest pepper producer where our country at the

fifth largest with 25, 672 metric tones of pepper production (Anita et aI., 2013). About

99% of the total area of Malaysian pepper is produced in Sarawak while other state that

also produced pepper is Johor, Malacca, and Sabah. Usually in Sarawak, pepper is

cultivated in small farms and it is important income sources of rural families. However,

profits from pepper crop depend on the production where high yield will contribute to high

income. Pepper price and price of inputs are some of market factors that affecting pepper

farming in Sarawak. Minister of Plantation Industries and Commodities, Datuk Seri

Douglas Unggah Embas has a strategy to farmers by introducing new incentives for pepper

farmers and new technology to increase pepper production (The Star, 2013).

1

Table 1.0: Characteristics of recommended Pepper Varieties in Sarawak (Adopted from AgricuJture Research Centre, 2006)

Variety Characteristics Kuching Semongok Emas Sernongok Arnan

Green berry yield 6-8 6-8 6-8 (kg/vine/year) % Driage ( conversion 33 31 33 ratio) to black pepper % Driage ( conversion 24 22 22 ratio) to white pepper

IWeight of 100 mature ..

13.8 15.6 15.7 green berries (g) Length of fruit spike (cm) 9.7 9.9 10.1 Chemical quality

3.5 5.4 % oleoresin

3.4% piperine 11.0 11.0 15.5

% volatile oil 2.8 3.0 3.8 % non-volatile oil 7.9 8.0 11.5 Harvesting rounds per 2 - 3 (more 2-3 (more season

4-6 uniform ripening) uniform ripening)

Susceptibility to:

a. Phytophthora foot rot disease Highly susceptible Susceptible Less susceptible

Highly susceptible b. Black berry disease Tolerant Tolerant Ic. Pepper weevil Less susceptible Less susceptible Less susceptible

In Sarawak, Kuching and Sarikei are two local varities of pepper that has been cultivated

since 1856 (Department of Ariculture, 2006). Kuching is the most extensively grown

cultivar in Sarawak. Moreover, it has vigorous growth and is high yielding. Sarikei is a

small stature plant with smaller leaves and has lower yield than Kuching. This variety also

has lost its popularity and no longer being cultivated. According to Research Division of

the Department of Agriculture (2006), new varieties are being developed and currently

being assessed under field conditions. Table 1.0 shows in year 2006, the recommended

cultivars are Kuching, Semongok Emas and Semongok Arnan.

2

Malaysian Pepper Board (MPB) is agency that responsible for processing and pepper

grading, promotion, quality improvement of pepper, product development of pepper and

other process related to pepper. Betong is one of the biggest areas of pepper farms which it

is the centre of production for the profitable creamy white pepper (The Star, 2013). To

increase the production of pepper, pepper planting areas will be expanded to 20, 000 ha.

According to Dato Sri Douglas Unggah Embas, pepper industry is expected to increase

export income by year 2020. He said several strategies need to be taken to achieve the

target including strengthen research and development activities, increasing productivity

and human capital development (New Sarawak Tribune, 2013). He also advices

smallholders to planting other crops such as rubber or oil palm to keep them afloat when

price ofpepper are down.

People are looking forward to produce a product that is environmental friendly as it is

among the major concern in today's world. In agricultural industry, researchers also try

hard to produce environmental friendly product. One of effort is by producing organic

product instead of using chemical. Biofertilizers are one of the essential components of

organic farming where people are concern on environmental pollution associated with

chemical use. It play important role in improving soil fertility which can sustain crop

production. Pollution and contamination of soil is largely has polluted water basins hence

destroyed microorganisms and other aquatic life. According to Sheraz et al. (2010),

biofertilizers should be recommended as supplement for maximizing the crop yield and

also for agro system stability.

3

Around 40% of chemical fertilizers are extensively used throughout the most of

agricultural in Asia which it consume the world's largest user of the chemical fertilizers. In

Malaysia, marketing strategy of biofertilizers is through niche markets such as

ornamentals, vegetables and forestry. But the scope of biofertilizers product seems to be

limited; for example rhizobial inoculums could only be applied to legumes (Khairuddin,

2002). For big plantation industry, biofertilizers products were used together with chemical

fertilizers to get good results.

1.1 Problem Statements

Chemical control by using inorganic fertilizers and pesticides has led to serious

environmental problems such as depletion of soil quality and health and emergence of

resistant pathogens. Only little work has been done in this biological control of disease in

vegetatively propagated crops (Anith et aI., 2002). Root rot disease by P. capsici has

caused high mortality of cuttings and cause economic losses. Other new diseases such as

stunted disease and anthracnose effect production of black pepper (Anandaraj & Sarma,

1995).

In Malaysia, farmers and smallholders received fertilizers subsidized by the government.

Currently, Malaysian government provides RM 500 million per year fertilizer subsidy

(Khairuddin, 2002). In the case, farmers often perceived that biofertilizers are more

expensive than chemical fertilizers and only certain nursery and farm operators use the

biofertilizers as they appreciate the benefits of it. Knowingly that chemical fertilizer has

been introduced in the industry, it is difficult to shift to biofertiIizer used and the

application on its own may not be sufficient in this high productivity agriculture

(Khairuddin, 2002).

4

Pusat KI.idmat Maklumat Akademik UNlVERSm MALAYSIA SARAWAK

" 1.2 Obj ectives

This project is aim to implement green agricultural practices to protect and conserve the

environment by using land for agricultural practices in sustainable way. The specific

objectives are:­

i. To test and evaluate the use ofbiofertilizer developed on black pepper.

u. To improve the quality of black pepper and increased yield.

5

2.0 LITERATURE REVIEW

2.1 Black Pepper

Black pepper is known throughout the world as it famous as king of spices; which it also

known as peppercorn. Botanical name of this black pepper is Piper nigrum Leonian (P.

nigrum L.). P. nigrum is the member of family Piperaceae and its genus has more than

1000 species (Ahmad et al.) 2012). Usually pepper plant was cultivated for its fruits to

produce black, white and green pepper. Many use of black pepper including medicine,

preservatives, human dietaries and also biocontrol agents. Black pepper has its value due to

its better pungency and aroma and also presence of piperine. Pepper is affected by several

diseases caused by bacteria, fungi, virus, mycoplasma and also by nutritional disorders. It

is been recorded that about 17 diseases in black pepper but the common diseases that

attacked black pepper are root rot disease caused by Phytophthora capsici. Usually, the

disease is undetected by farmers until the upper part of pepper vine shows symptoms of

leaf yellowing, wilting and dropping (Ton & Buu, n.d). This disease may cause severe

losses ofcrops which it can be controlled by chemical and biological control.

Black pepper is a viny perennial plant producing berry-like fruits; the dried ripe or unripe

berries become black pepper and soaking to remove outer skin of berries will become

white pepper. It does not tolerate excessive heat and dryness. Leaves of black pepper are

green in colour and thick with ovate shape. Although black pepper grows in almost all

types of soil, but it thrives well in loose and well drained soil under humid climate with

rainfall of 100 to 250 cm.

6

Although India is the largest producer of pepper crop in the world, productivity of the crop

is low. This is due to several factors such as poor genetic potential of the vines, losses

caused by pests, disease and drought, and non availability of quality planting material of

improved varieties (Sivaraman et al., 1999).

2.1.1 Pepper Cuttings for Propagation

Selection criteria for planting materials is by cultivate only varieties of pepper

cuttings. Cuttings for propagation should be taken from vigorous which are below the

node and 20 inches or more in length. Three types of aerial shoots was developed from

black pepper vines which is primary stem with long internodes, runner shoots which

originate from the base of the vine and fruit bearing lateral branches. Conventionally,

black pepper was propagated through cuttings with two to six nodes for field

plantation; as it is traditionally propagated by stem cuttings (Abbasi et aI, 2010).

There are few methods for propagated the cuttings; traditional method, rapid

multiplication method, trench method, and serpentine method (TNAU, 2013).

Production of rooted cuttings for traditional method is by kept coiled of runner shoots

on wooden pegs at the base of the vine to prevent the shoots from coming in contact

with soil. Rapid multiplication method was an efficient propagation technique was

developed at Sri Lanka but has been modified for adoption in India (IISR, 2014).

M odification of the technique is for quick and easy multiplication of black pepper

vines. Another method is trench method, a simple, cheap and efficient technique for

propagation from single nodes of runner shoots (IISR, 2014). Single node of 8-10 cm

length with their leaf intact, are to be planted in the bags in such a way their leaf axil is

above the potting mixture. Serpentine method is the cheaper propagation technique

7

where rooted black pepper cuttings are planted which it will serve as mother plants

(IISR,2014).

2.2 Biological Control

Biological control is defined by used of organisms, genes or gene products to regulate a

pathogen and also can retard infection and minimize plant systems for self-defence

(Thankamani et al., 2004). The biological control agents such as Trichoderma sp., V AM,

and plant growth promoting rhizobacteria is used for preventing the disease in black

pepper nursery as well as in the field. Commonly used as bio agent on black pepper is

Trichoderma sp. which probable mechanism of disease control by it is hyper parasitisation,

production of volatile and non-volatile antibiotics to suppress soil borne pathogens and

starving the target pathogen by taking the available nutrients (Thankamani et al., 2004).

The utilization of manure treated with Trichoderma species can reduce the population of

soil-borne fungi and nematodes and incidehce of foot rot (Ton & Buu, n.d).

Biological control is the best alternative method as a strategy for sustainable disease

management although chemical control is more effective (Paul et al., 2005). Moreover,

chemical control contributes to environmental hazards which involved high cost for the

chemical pesticides. As chemical control has a negative effect on environment, concerns

on this chemical applications in agricultural activities has encouraged scientist to search for

other methods. Currently, biological control is the best method which requires a simple

screening for biocontrol agents but it is not always sufficient to find effective biocontrol

agents (Chang et al., 2001). By using bioassays, several tests must be conducted on plants

after screening antagonistic agents. It is critical to understanding the mechanism of

8

biocontrol for the improvement and effectiveness of the biocontrol agents used (Paul et ai.,

2005).

Biological control can be divided into three main techniques; classical contro~

augmentative control and conservation control (Bale et al., 2008). Classical or sometimes

described as inoculative control usually used against exotic pests that has become

established in new countries of the world. For augmentation control, it refers to all forms

of biological control which usually require the commercial production of the released

agents. This type of control used natural enemies that are periodically introduced. Another

type of biological control is conservation control that usually against native pests by using

indigenous predators and parasitoids (Bale et al., 2008).

Sixty years ago before chemical pesticides was used in a large scale, biological control was

the main pest management method to prevent and reduce pests, covering animal, weeds

and pathogens (Bale et aI. , 2008). How~ver, around 1950, biological method became

redundant where almost pest be able control by the newly discovered pesticides (Bale et

al., 2008). Using pesticides has led to many effects not only to plants but also to the

environment. According to Bale et al. (2008), planted crops has turn into 'incubator plants'

which this cops unable to survive without frequent application of the pesticides. As

chemical pesticides are no longer seen as solution for sustainable pest management, there

is need to restore previous used of biological control; natural, classical, inundative and

conservation biological control. Further, it also needs to invest in the development of new

crop cultivars. The re-introduction of the pest management is successful by reducing the

application of fertilizer used although it produce lower yield due to lower inputs of

fertilizers or pesticides (Bale et al., 2008).

9

" 2.3 Blofertilizers

Products that contain of different types living cells of microorganism are defined as

biofertilizers. Microorganisms include bacteria, fungi and green algae can be used as

biofertilizers. This microorganism is able to mobilize the nutritionally important elements

through biological processes.

There are several types of biofertilizers which is nitrogen, phosphorus, and compost

biofertilizers (Adnan, 20 I 0). Nitrogen biofertilizers helps to determine nitrogen level in

soil as it is necessary component that is used for the growth of the plant. While for

phosphorus biofertilizers, it supply phosphorus to the soil and make the soil get the

required amount of phosphorus. By using animal dung, compost biofertilizers enrich the

soil with useful microorganisms and nutrients contain in the animal waste. This animal

waste converts into biofertilizers by breaking down the waste.

Biofertilizers has many advantages than chemical fertilizers as it not beneficial for the

plants. Chemical fertilizers usage in agricultural have polluted the environment by

releasing the harmful chemicals. Growth rate of plants is increasing when using

biofertilizers because it contains natural components that do not harm the plants and

environment. By using biofertilizers, it will improve plant and also soil health as they act

as plant strengtheners, phtostimulators, plant health improvers, and have the potential to fix

nitrogen (LawaI & Babalola, 2014). But the most important is biofertilizers are cost

effective where smallholders farmers can afford to use. While for the environment,

biofertilizers are environment friendly and protect environment from the pollutants.

Fertility of soil will retain which it is beneficial for plants hence plants are protected from

getting any diseases (Amna, 2010).

10

/

2.3.1 Previous Study on Biofertilizers in Black Pepper

Since the demand for the spice in the market has increase, biofertilizers has been used

for meeting the nutrient needs of pepper and help to improve yield and also the quality

of crops. Consumers are willing to pay a premium price for organic products against

conventional products (Nybe & Stephen, 2003). A scientific research has been

conducted on application of biofertilizers in black pepper. According to Nybe &

Stephen (2003), treatments involving complete organic and biofertilizer, organic and

inorganic combinations exhibit higher values ofall soil nutrients.

Kandiannan et al. (2000) has conducted an experiment on growth and nutrient content

of black pepper as influenced by inoculation with biofertilizers. It shows that black

pepper responded well to the combined inoculation with biofertilizers (Kandiannan et

ai., 2000). Results that have been recorded showed growth was significantly higher

when inoculated biofertilizers compared with individual inoculation and control; this

maybe due to the collective effect of biofertilizers. More yield were produced when

plants was inoculated with both VAM and Azospirillum than single inoculation. This

combined inoculation also fixed more nitrogen than singly inoculated plants in pearl

millet (Tilak, 1995).

11

2.4 Plrytopthora capsici

.,;

Phytopthora capsid Leon is a soil borne pathogen that found in black pepper. Phytopthora

infections can be classified into aerial and soil based (root rot). Aerial infections can occur

on the stern, foliage, runner shoots, spikes and branches (Anandaraj & Sarma, 1995). It can

cause die-back, defoliation, spike shedding, blight, and death of plants (Hua, n.d). Also, the

disease can be spread by contact with soil particles or from water run off. Disease caused

by P. capsid will reduce transportation of water and nutrition as roots become rotted.

Rotting of collar and roots will cause plant death. Visible changes on black pepper due to

P. capsid infection can be seen as the infection usually starts at the collar region of the

vine (Hua, n.d). The branches droop, the underground stem has brownish black lesion and

the leaves progressively tum yellow, wilt and defoliated (Hua, n.d). However, the disease

disappeared when black pepper grows intercropped between rows of cacao, coffee or other

fruits trees (Noveriza & Quimio, 2004)". Previous study has proved that Trichoderma are

the most potential agent for biocontrol which inhibited the production of sporangia and

zoospore ofP. capsid.

Farmer's should adopt an integrated disease management system to prevent disease

occurred in the farm or gardens. The most important is the gardens should have good

drainage system and discouraged sharing farrrt tools and foot wares between gardens.

Pepper cuttings should be obtained from root rot-free gardens. This practice is to avoid

introducing the disease in new area. Pruning oflower branch ofthe pepper plant need to be

done to prevent the branches come in contact with the soil. It needs to be prune when the

lower branches are about 30 cm above the ground (Hua, n.d).

12

2.4.1 Phytophthora blight

Phytophthora blight or crown blight of peppers also caused by the fungus-like

organism Phytophthora capsid. P. capsid is a group of organisms that sometimes

referred as 'water molds' and it is an oomycetes which produce several types of spores

(Louws et al., 2008). Other names for this disease are damping off and Phytopthora

root rot, crown rot, and stem and fruit rot. The name of the disease can apply since all

parts of the pepper plant are affected. Phytopthora blight disease has been spread

sporadically for more than 40 years in New York (Thomas, 1989).The disease is one

ofthe most important diseases worldwide. It can affect plants at any growth stage, and

the pathogen can also cause crown, leaf and fruit blight, wilting of the whole plant and

dark purplish discoloration of the stem (Akgul & Mirik, 2008). During heavy rainfall,

it can become a serious problem as the pathogen can spread rapidly through the crops

which will result in severe losses within a short time (Sally et a/., n.d).

When there is excess in soil moisture, fungus P. capsid can occur naturally in most

soil and infect pepper plant and also other crops. It infects the crops at most stages of

growth. Good cultural practices can prevent the disease to be occurred in the field. The

most important attention to be given is avoiding poorly drained fields for growing the

crops. To provide better soil drainage, plant the crop on a ridge or dome-shaped beds

(Thomas, 1989). Other cultural practices can be used is practice crop rotation with

crops other than tomato, eggplant and cucurbits for a minimum two years but

preferably four years. If the crops have been infected with P. capsid, the infected

plants must be eliminating immediately to limit further spread ofthe disease (Louws et

al., 2002).

13

2.S Previous Study using Bacteria Burkholderia unamae and Enterobacter clocae

2.S.1 Burkholderia unamae PI0

Burkholderia bacteria are human and plant pathogens as well as environmentally

important bacteria. Burkholderia unamae is a non pathogenic species although its

species Burkholderia is investigated to involve in pathogenesis. It is single-celled

prokaryote microorganisms that have a wide range of shapes; from spheres to rods and

spirals. B. unamae belong to species proteobacteria that exist in every habitat on Earth

such as in soil, water, live on plants and animals bodies (Surhone et al., 2011).

Burkhoideria and related bacteria have been found at soils of all temperature including

Arctic soil of 7°C (Microbe Wiki, 2010). It is shown that Burkholderia is rich in

nitrogen fixing and to serve as plant growth promoting factors. This microorganism

was isolated from rhizopheres and plants growing in soil with pH value range 4.5 - 7.1

(CabaUero-Mellado et al., 2004).

B. unamae is recently shown rich in N2-fixing that is associated with plants. Present

study of a polyphasic taxonomic study which includes new isolates that recovered

from rhizopheres, rhizoplanes or internal tissues of maize, sugarcane and coffee plants

(Caballero-Mellado et al., 2004). The isolates of B. unamae grew and showed the

ability to reduce acetylene in N-free, semi solid BAz medium as wee as with succinate

and propionate as carbon source (CabaHero-Mellado et al., 2004). It can be

differentiated with B. tropia which is the most closely related N2-fixing species. The

differentiation is by its inability to assimilate p-gentiobiose and ribose. Moreover, B.

unamae can be differentiated with B. sacchari by its ability to assimilate cellobiose,

rhamnose and trehlose (Caballero-MeUado et aI. , 2004).

14

2.5.2 Enterobacter cloacae Pll

Enterobacter cloacae belong to the family Enterobateriaceae is a gram negative

bacterium, facultatively anaerobic, have a rod-shaped cells and motile (Charles et ai.,

1987) and some of which are encapsulated. As facultative anaerobes, some

Enterobacter bacteria ferment both glucose and lactose as a carbon source. This

bacterium can be found on human skin and plants as well as in soil, water, sewage,

intestinal tracts of humans and animals. In industrial use, E. cloacae have been used in

bioreactor-based for biodegradation and as biological control of plant diseases. This

species are widely encountered in nature and they also can act as pathogens in plants

and insects (Mezzatesta et ai., 2012). They are found in soil and sewage which these

microorganisms are saprophytic in the environment. Enterobacter bacteria can infect

human which some of the symptoms include bacteremia, lower respiratory tract

infections, skin infections, soft tissue infections and urinary tract infections (Grimont

& Grimont, 2006). The bacterium which is called ' yellow pigmented Enterobacter

cloacae' until 1980 is survived when the contaminated powered formula is heated and

prepared.

Rhizopus spp. is aggressive parasites, causing major losses in postharvest fruit of

peach fruit (Prunus persica (L.) Batsch). This Rhtzopus spp. is difficult to control but

presently it being control by accomplished with fungicides, hydrocooiing, hot water

dips, high temperature storage and refrigeration (Charles et ai. , 1987). When peach

fruits were treated with E. cloacae, R. stolonifer infections were inhibited and mostly

Rhizopus lesions developed after eight days (Charles et aI., 1987).

15