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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).
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