BIBLIOGRAPHY RACHEL K. TUDAYAN. November...
BIBLIOGRAPHY
RACHEL K. TUDAYAN. November 2006. Characterization of the Pathogens
Associated with the Disease Complex of Mondo Grass (Ophiopogon jaburan (Sieb.)
Lodd.). Benguet State University, La Trinidad, Benguet.
Adviser: Janet S. Luis, Ph.D.
ABSTRACT
The characterization of the pathogens infecting the mondo grass was done based
on symptoms, signs and cultural and morphological characteristics.
The disease symptoms observed includes leaf spots, blights, yellowing and
necrotic lesions. The pathogens appeared either singly or interacting together resulting in
a disease complex.
Three organisms were identified in the disease complex which include the fungus
Fusarium sp., the bacterium Xanthomonas sp. and the nematode Aphelenchoides
fragariae.
TABLE OF CONTENTS
Page
Bibliography …………………………………………………….
i
Abstract ………………………………………………………..
i
Table of Contents ……………………………………………...
ii
INTRODUCTON ……………………………………………..
1
REVIEW OF LITERATURE
The Host ……………………………………………………..
4
Disease ………………………………………………………
5
Diseases Caused by Bacteria ………………………………..
6
Diseases Caused by Fungi …………………………………..
8
Diseases Caused by Nematode ……………………………...
9
MATERIALS AND METHODS
Collection of Diseased Leaves ……………………………….
13
Description of Symptoms and Microscopic Observation
of Signs ……………………………………………………..
13
Identification of the Isolated Fungus ……………………...
13
Isolation …………………….…………………………...
13
Identification ……………………………………………
14
Identification of the Isolated Bacterium ……………………
14
Isolation ………………………………………………..
14
Identification …………………………………………..
15
Identification of the Foliar Nematode ……………………….
15
ii
Isolation ………………………………………………..
15
Identification …………………………………………..
15
Staining of plant tissues ……………………………...
16
Data Gathered ……………………………………………….
16
RESULTS AND DISCUSSIONS
Fungal Component of the Disease Complex ………………..
17
Disease symptoms and signs …………………………
17
Cultural characteristics ……………………………….
18
Morphological characteristics ………………………..
19
Bacterial Component of the Disease Complex ……………...
20
Disease symptoms and signs ………………………...
20
Cultural characteristics ……………………………….
22
Growth in differential media …………………………
23
Gram stain reaction ……………………………..........
24
Potassium hydroxide (KOH) test …………………….. 25
Nematode Component of the Disease Complex ……………. 26
Disease Symptoms and Signs …………………………... 26
Morphological Characteristics ………………………….. 28
Staining reaction ………………………………………... 29
SUMMARY, CONCLUSION, RECOMMENDATIONS ……. 32
LITERATURE CITED ………………………………………… 35
APPENDICES ………………………………………………... 38
iii
INTRODUCTION
Plants dominate the earth and are considered the most valuable things in
the natural world, affecting all aspects of human lives either directly or indirectly.
Human and animal lives depend mainly on the oxygen that these produce and
recycle and the ability to produce organic matter. Plants are the major source of
food and other basic necessities such as medicines, oils and fibers.
Plants contribute to the enhancement of the environment. These are used
in landscaping either as cover crops, shades and decorations. These aesthetic
purposes according to Rimando (2001), give warmth and color, peace and
serenity to the work area.
In the past centuries, humans learned to settle down and started cultivating
and domesticating plants and animals as sources of food supply. All over the
world, most of the fertile lands are already converted for agricultural use. In
reality, the food supply of human and animal population is determined by the
growth and productivity of plants.
Plant growth and productivity are, however, affected by several factors
one of which are diseases. The interaction between the plant and pathogenic
organism affects the food supply. Nowadays, a constant war occurs between the
farmer and these pathogens. The management of these diseases brings about
financial losses in terms of the use of chemical pesticides. These affect not only
the quantity but also the quality of the plant product causing considerable
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economic impact. On the other hand, symptoms of the disease in certain plants
may occur singly or in characteristic combinations. The causal organism may
also be due to one or more agents.
Mondo grass (Ophiopogon jaburan (Sieb.) Lodd.) which was introduced
in the Philippines during the early 1990’s (Madulid, ) is being used as a cover
crop or as a border plant in some of the localities within La Trinidad, Benguet and
Baguio City. Some are found to be planted along the roadside and or are
integrated with other ornamentals in a quadrangle.
It is observed, however, that most of these plants are attacked or infected
with disease. Symptoms of leaf spots, yellowing and browning of the green
leaves are observed.
It is therefore apparent to verify and identify the diseases of mondo grass
to determine if these pose a threat to other important crops such as vegetables,
fruits and ornamentals within the locality.
To determine the range of diseases attacking the plant and to fully
understand the nature and significance of these diseases, this study aimed to:
1. Describe the symptoms of the disease complex observed in mondo
grass;
2. Isolate and describe the pathogens associated with the disease
complex; and,
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3. Identify and characterize the pathogens attacking mondo grass based
on their cultural and morphological characteristics.
The study was conducted at the Plant Pathology Service Laboratory,
College of Agriculture, Benguet State University, La Trinidad, Benguet
from March to October 2006.
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REVIEW OF LITERATURE
Host
Ophiopogon or mondo grass is a perennial crop belonging to the
monocot group. It has the following plant classification
(http://plants.usda.gov.java/profile):
Kingdom …………………….Plantae – Plants
Subkingdom ………………..Tracheobionta – Vascular plants
Superdivision ……………..Spermatophyta – Seed plants
Division ………………….Magnaliophyta – Flowering plants
Class ……………………Liliopsida – Moncotyledons
Subclass ……………….Liliidae
Order
…………………Liliales
Family ……………….Liliaceae – Lily family
Genus ………………Ophiopogon Ker-Gawl
Species …………..Ophiopogon jaburan (Sieb.) Lodd.
Ophiopogon is native to the Honshu, Shikoku, and Kyushu,
provinces of Japan. It grows in woods and scrub at low altitudes,
flowering in July-August. This plant has thickened tuberous roots,
spreading by stolons to form loose patches. The leaves are narrow at 2-3
mm width commonly planted as ground cover or as low-maintenance
grass substitute (Phillip and Rix, 1996).
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Mondo grass is one closely related, grass-like groundcover. The
most frequently used species is Ophiopogon japonicus, which has several
popular varieties. These are generally drought-tolerant as well as disease-
and insect-resistant. Mondo grass is used for edging borders and in places
where minimal maintenance is required. Variegated selections can be
used as accent plants to add contrast and color
(www.ctahr.hawaii.edu/oc/freepubs/pdf/of.28.pdf).
The Disease
“Since it is not known whether plants feel pain or discomfort, as
plants do not speak or otherwise communicate to us, it is difficult to
pinpoint exactly when a plant is diseased” (Agrios, 1997).
To cause a disease, a pathogen must find a suitable host plant, pass
through the external protective layers of the host, and gain access to the
nutrients that it requires for its own growth and development (Dickson,
2003).
The emerging disease is dessiminated and spread by air, water,
insects and other vectors and most especially dessiminated by humans
through the tools, machineries and infected planting materials (Agrios,
2005). International trade also results in all sorts of plant materials being
moved from one place to place. Pathogens are often imported and become
established in countries where the conditions for disease development
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maybe more favorable than in the country of origin (Lucas, 1998). The
movement of plant and plant products is also an important source of new
diseases.
Diseases Caused by Bacteria
Bacteria are the most widely distributed, the simplest in
morphology, the smallest in size and the most difficult to identify. They
are prokaryotic and seldom photosynthetic (Benson, 1998).
There are a number of bacterial diseases of plants (Mount and
Lacy, 1982; Circrolo, Collmer and Gillaspie, 1987; Billing, 1987). Of the
1600 known bacterial species, 100 cause diseases in plants (Dickson,
2003). Most plant pathogenic bacteria are rod-shaped except
Streptomyces which is filamentous. Furthermore, most of the plant
pathogenic bacteria have delicate thread-like flagella, considerably longer
than the cells on which they are produced (Agrios, 2005).
Bacteria utilize a range of strategies for entry into plants. The leaf
surface can support large populations of many bacterial species that may
either be present in the air, splashed or carried by insects. These bacteria
can multiply on the surface to form microcolonies or larger aggregates
which enter into the plants through stomata, natural openings and wounds,
with the help of water droplets formed on the leaf surfaces. In some
Xanthomonas bacteria, they actively invade the plants through the
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hydathodes, the structure containing water pores located at leaf margins.
Under suitable conditions, copious amounts of fluid are exuded through
the hydathodes that are collected as guttation water drops around leaf
margins. The bacteria move chemotactically towards these drops, then
later is drawn back into the hydathodes carrying the bacteria with them in
the vascular system (Dickson, 2003.)
Bacteria can not enter plants via intact cuticles. Their entry is
through wounds and natural openings such as stomata or lenticels. The
presence of water is necessary to enable the pathogen to multiply and
move to a point where entry is possible. Inside the plant, they first
multiply in the intercellular spaces and/or the xylem and produce
pectolytic enzyme which causes cell death and the cells are then invaded
(Manners, 1993).
Agrios (2005) described the symptoms of bacteria such as leaf
spots and blights; soft rot of fruits, roots, and storage organs; wilts;
overgrowths; scabs and cankers. The most common type of disease in
plants are those that appear as spots of various sizes on leaves, stems,
blossoms and fruits. In some bacterial diseases, the spots continue to
advance rapidly and are then called blights.
In monocotyledonous plants, bacterial spots appear as streaks or
stripes. In humid weather conditions, infected tissues often exude masses
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of bacteria that spread to new tissues or plants and start new infections.
Almost all bacterial spots and blights of leaves, stems and fruits are caused
by bacteria belonging to the genera Pseudomonas and Xanthomonas.
Diseases Caused by Fungi
Fungi are usually filamentous, eukaryotic and spore-producing
organisms that lack chlorophyll. The cell walls are made up of chitin
combined with other complex carbohydrates and cellulose. All species of
fungi are either saprobes or symbionts. As symbionts, they may provide
certain benefits to their host, or may parasitize their host (Moore, et. al,
1995). The unit structure is the hypha, a branched filament consisting of a
row of cells with or without coenocytic structure. Reproduction usually
involves the production of unicellular or multicellular spores by sexual or
asexual processes.
Fungi may cause a very wide range of types of plant disease. More
than 10,000 species of fungi can cause disease in plants (Agrios, 2005). In
plant pathogenic fungi, there are sub-specific entities which attack
different host genera. All plant pathogenic fungi make use of extracellular
enzymes which they secrete at most stages of their attack on their hosts
(Manners, 1993).
Fungi and oomycetes are generally dispersed as spores that are
either deposited in the soil or transmitted through the air. Once the fungus
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adheres and germinates within a suitable host, it grows into the plant and
obtain its nutrients for its growth and development. It has the capacity to
do this either directly through the cuticle or grow towards the natural
opening or wound sites and enter through these (Dickson, 2003).
Fungi can cause local or general necrosis on plant tissues, and they
often cause reduced growth of plant organs or entire plants. Some of the
most common necrotic symptoms are leaf spot, blight, root-rot, damping
off, anthracnose, soft rot and dry rot. In many diseases, the fungal
pathogen grows or produces various structures on the surface of the host
which is of particular importance including mycelia, sclerotia,
sporophores, fruiting bodies and spores (Agrios, 2005).
Diseases Caused by Nematodes
Nematodes belong to the kingdom Animalia. Nematodes are
cylindrical worms, with a body shape commonly described as filiform or
having the shape of a thread. The name “Nematoda” is derived from the
Greek word nema, which means thread. They are aquatic animals that
inhabit oceans, seas, freshwater courses, body fluids, and in the film of
water present between soil particles and in plant which is of particular
importance.
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Plant parasitic nematodes are present in almost every type of
habitat with the limiting factors being moisture and food for survival.
These are obligate feeders on plants and can only obtain nutrients for
development and reproduction from the cytoplasm of living plant cells
(Perry and Wright, 1998). They constitute about 20% of the described
species (4,000 among 20,000) within the phylum Nematoda and are
included in the classes Chromadoria and Enoplea (Ferraz and Brown,
2002).
Nematodes are considered the most successful pseudo-coelomate
animals. This is due to their highly integrated body walls (cuticle,
epidermis and longitudinal muscles) that permit very efficient locomotion
(Lorenzen, 1985). Another contributing factor is the ability of some
nematodes to enter a state of anabiosis which permits long-term survival
under extremely harsh conditions. Nematodes have adapted to and are
capable of surviving a variety of extreme environmental/physical stresses
and even possess specialized developmental stages in order to do this
(Perry and Wright, 1998).
Female nematodes lay about 300-500 eggs. Depending on the
climate, the availability of hosts and the duration of each life cycle of the
particular nematode species may have from two or more than a dozen
generations per year.
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Symptoms caused by nematodes on roots may appear as root
lesions, root knots or root galls, excessive root and accompanied by plant
pathogenic or saprophytic bacteria and fungi as root rots. Certain species
of nematodes invade aboveground portions of plants. They cause galls,
necrotic lesions and rots, twisting or distortion of leaves and stems, and
abnormal development of the floral parts (Agrios, 2005).
Plant parasitic nematodes interact with many soil inhabitants
(fungi, micro-arthropods and free-living nematodes) and their soil
environment is significantly affected through human intervention with
agricultural procedures (Ferraz and Brown, 2002). Since most of the
nematodes live and operate in the soil where they are surrounded by many
soil inhabitants, there is a great interrelationship between the nematodes
and other plant pathogens. In many cases, an association develops
between nematodes and the other pathogens. Nematodes then become a
part of an etiological complex resulting in a combined pathogenic
potential that sometimes appears to be greater than the sum of damages
that either of the pathogens can produce individually (Agrios, 2005).
In the Philippines, published records show that nematodes are
prevalent in the Philippine soils. The plant parasitic forms apparently
have received much attention since they are frequent associates of plant
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diseases and are thus potential threat to agriculture (Castillo and Reyes,
1972).
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
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MATERIALS AND METHODS
A. Collection of Diseased Leaves
Sample leaves of infected mondo grass were collected, placed in
transparent polyethylene bags and brought to the laboratory for further diagnosis.
Samples were obtained from different locations in La Trinidad, Benguet and
Baguio City.
B. Description of Symptoms and Microscopic Observation of Signs
Symptoms of the diseased plants were described. To observe the signs,
thin sections and scrapings from infected fresh materials were mounted into a
drop of water on a glass slide then covered with cover slip and were studied under
the binocular compound microscope. All observations were recorded. Books,
journals and other reference materials on fungi, bacteria and nematodes were used
in characterizing the symptoms manifested by the mondo grass leaves and the
signs observed.
C. Identification of the Isolated Fungus.
Isolation. The specimens were washed with running tap water to get rid
of dirt, were cut into small sections about 2-5 sq. cm. then each piece of cut
tissues were disinfected with 10% sodium hypochlorite for 1-2 minutes. After
disinfection, the cut sections were rinsed with three changes of sterile distilled
water and were then blot-dried on a sterile tissue paper. Specimens were
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aseptically placed equidistantly on plated potato dextrose agar (PDA) then were
incubated for 3-5 days or more at 28-30°C. The culture medium was prepared
following the standard procedures. Pure culture of the fungal growth were re-
isolated in PDA slants, stored at 5°C which served as stock culture.
Identification. The cultural characteristics such as color and presence of
pigmentation were noted and recorded. The microorganisms were examined
under the electric binocular microscope taking note of their morphological
characteristics such as kind of mycelia produced, spore morphology and other
structures.
D. Identification of Isolated Bacterium
Isolation. The collected infected leaves were washed with tap water to
get rid of the surface dirt, disinfected with 10% sodium hypochlorite and were
finally rinsed with three changes of sterile distilled water. Leaf samples were
placed in a water blank and were macerated with a flamed glass rod to hasten the
oozing of bacterial cells. A loopful of the resulting solution was streaked onto
previously prepared nutrient agar (NA) which was prepared following the
standard procedures. The plates were incubated at 28-30°C for 24-72 hours. To
obtain a pure culture, a single colony of the bacterium was reisolated separately
into another plated NA. Stock cultures were kept on agar slants at 5°C.
Identification. To determine the genus of the isolated bacterium, the
following tests were done:
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1. Growth on various media
a. Yeast Extract Dextrose Agar (YDCA)
b. King’s Medium B Agar (KMBA)
c. Nutrient Glucose Agar (NGA)
d. Sucrose Peptone Agar (SPA)
2. Gram stain and potassium hydroxide (KOH) test
E. Identification of the Foliar Nematode
Isolation. Plant tissues were cut into longitudinal sections and were
placed in a glass Petri dish containing distilled water. The set/up was allowed to
stand for minutes to allow the nematodes to migrate and be released from the
damaged tissues. With the aid of a dissecting microscope, the nematodes were
then collected using an improvised hand picker (stick broom).
Identification. The mouth, stylet, tail and other distinguishing structures
of the isolated nematode were examined under the binocular electric microscope.
Identification was done with the aid of books, journals and other related available
references.
Staining of plant tissues. In order to observe the nematodes inside the
leaves, plant tissues were stained (modified technique) by cutting the leaves into
small sections about 2-3 sq. mm. then were soaked in a carnoy’s solution for a
few days until the chlorophyll pigmentation was removed. When the leaves
became clear, acid fuchsin (1 ml stock solution and 30 ml water) was added and
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was left for at least a day to be enable the stain to be absorbed by the nematode.
After staining, the leaves were rinsed in running tap water to remove excess stain
and were soaked and covered with pure glycerin.
Data Gathered
The following data were gathered:
1. Symptoms of the disease complex. These refer to the manifested
abnormalities on the diseased leaves of mondo grass.
2. Signs of pathogens involved in the disease complex. These refer to
the specific structures of the fungus, bacterium and nematode that
were freshly scraped or cut into thin sections from the diseased tissues
and observed under the binocular compound microscope.
3. Cultural and morphological characteristics of the isolates. These refer
to the color/pigmentation in PDA of the fungal isolate and gram stain,
KOH reaction and colony appearance in NA and other selected
differential media of the bacterial isolate.
4. Identification of pathogens involved in the disease complex. This was
limited to the genus level for the fungal and bacterial components of
the disease complex and species level for the nematode component.
5. Photodocumentation
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RESULTS AND DISCUSSION
Fungal Component of the Disease Complex
Disease Symptoms and Signs
Sample leaves exhibited circular to oval dark round spots on the leaves
surrounded by a definite yellow margin (Plate 1a). Other leaves showed blights
starting from the tip of the leaf moving downward (Plate 1b) . Other symptoms
observed showed association with other bacterial and nematode symptoms like
yellowing and necrotic spots.
a
b
Plate 1. Symptoms manifested by the suspected fungal component of the
disease complex appearing as leaf spot (a) and leaf blight (b)
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Plate 2. Signs observed under the compound microscope appearing as
septated boat-shaped numerous macroconidia with
microconidia
The microscopic examination of slide mounts prepared from thin sections
showed boat- or banana-shaped macroconidia with 4 to 6 septations. Numerous
microconidia were noted which imply the pathogenicity of the isolate (Plate 2).
Cultural Characteristics
The isolated fungus produces dense and compact light pink mycelia on
PDA. A pink-orange pigment is produced at the bottom of the plate but does not
diffuse throughout the medium (Plate 3).
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a b
Plate 3. Pure culture of Fusarium sp. with light pink mycelia at the top
(a) and pink-orange pigmentation at the bottom of the plate(b)
Morphological characteristics
Microscopic examination revealed slightly curved, banana-shaped
macroconidia with 4 to 6 septations. The septated mycelia are thick-walled.
There were numerous short and stout microconidia observed (Plate 4).
It should be noted that the type of macroconidia and microconidia
observed from the pure culture in PDA are exactly similar to those observed from
the fresh thin sections from diseased mondo grass leaves.
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Plate 4. Macroconidia and microconidia from the PDA culture of the
fungus
Bacterial Component of the Disease Complex
Disease Symptoms and Signs
Infected leaves manifested different kinds of symptoms appearing as leaf
spot and blight. Some symptoms have small dark brown lesions which start
from the leaf margin and moving longitudinally along the margin. Other
symptoms appear as spots of various sizes on the edge of the leaves then continue
to advance towards the healthy portion resulting in blights. Some symptoms
first appeared at the tip of the leaves then moving downward (Plate 5a). During
the rainy season, the symptoms had a yellowish and water-soaked appearance
(Plate 5b).
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a
b
Plate 5. Symptoms manifested by the suspected bacterial component of
the disease complex appearing as small dark brown lesions (a)
and yellowish water-soaked appearance (b)
Plate 6. Bacterial streaming from cut tissues
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The microscopic observation of the thin sections prepared from the
diseased leaves showed massive bacterial streaming especially those taken from
the lesions with dark brown discoloration (Plate 6). Bacterial streaming is one
property that is synonymous to phytopathogenic bacteria which are motile due to
the presence of flagella. Motility can be readily observed especially if the
material is fresh (Goszczynska, et. al, 2000).
Cultural characteristics
In NA, 72-hour old cultures of the suspected bacterium had yellow
colonies (Plate 7). The yellow color looked like an egg white with egg yolk at the
middle when seen through light which is a typical indication that the organism
involved belongs to the genus Xanthomonas (Lando, personal communication).
Plate 7. Bacterial isolate in NA 72 hours after isolation
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a b
c d
Plate 8. Bacterial isolate in differential media appearing as dark
yellow in YDCA (a) and light yellow in KMBA (b) SPA
( c) and NGA (d)
Confirmatory identification in differential media revealed dark yellow
colonies on YDCA and light yellow color on the other media (Plate 8). These
characteristics confirm the description of Schaad and Stall (1998). The colonies
are mucoid, convex and shiny. The yellow membrane-bound pigments are
brominated arylpolyne esters (xanthomonadins).
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Gram Stain Reaction
Under the oil immersion objective (OIO), the stained bacteria are rod-
shaped and appear pink in color which indicates that these absorb the color of the
counterstain (safranin) thereby are classified as Gram-negative (Plate 9a.) Most
phytopathogenic bacteria are Gram-negative and rod-shaped (Goszczynska, et. al,
2000). Bacteria that cause leaf spot or blight are often Gram-negative and may
belong to Pseudomonas or Xanthomonas (Agrios, 2005).
a b
Plate 9. Rod-shaped Gram-negative bacterial cells from smear prepared
from the NA culture (a) and mucoid thread produced from the
KOH test done on slide suspension of the suspected bacterial
isolate (b)
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Potassium Hydroxide Solubility (KOH) Test
The preliminary diagnostic identification of a bacterium using
potassium hydroxide (KOH - 3% aq., w/v) test showed a mucoid thread produced
indicating that the bacterium is Gram-negative (Plate 9b).
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Nematode Component of the Disease Complex
Disease Symptoms and Signs
The above-ground symptoms observed vary especially on the leaves.
These appear as necrotic lesions then advance towards the healthy leaves where
lesions coalesce. The affected tissues turn brown and sometimes discolored. Just
above the ground, the leaves appear yellowish turning brown with time causing
an early senescence of the leaves (Fig 10). Other symptoms manifested are leaf
blotching and yellowing while others are similar to the symptoms manifested by
the fungus and bacterium.
These observations indicate the presence of the three pathogens in the
disease complex. The sample plants collected showed that most often, these
organisms are all present at a time when examined under the microscope (Fig 11).
Frequently, nematodes facilitate the entry and establishment of other plant
pathogens. They may either have an “interrelationship” and “interaction” with
bacteria, fungi and viruses (Lopez et. al., 2004).
In 1892, interactions of nematode with other plant pathogens had already
been recorded. All plant pathogenic nematodes cause wound in plants by
puncturing, rupturing or by separating cells thereby introducing or aiding the
entry of other pathogens already present on the plant surface. Chen et. al. (2004)
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Plate 10. Symptoms manifested by suspected nematode component
of the disease complex appearing as yellow to brown
discoloration starting at the base of the leaves.
cited some reports on nematode-fungi interactions like: nematode with Fusarium
oxysporum and other Fusarium spp., Verticillium spp., Pythium spp., and
Cylindrocarpon spp. Bacteria-nematode interactions are also known to occur:
like nematode with Clavibacter spp., Pseudomonas spp. and Agrobacterium spp.
For viruses, the nematode vector belongs to the genera Xiphinema, Trichodorus
and Longidorus.
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a
c
b
Plate 11. Disease complex in the mondo grass as indicated by the
presence of hypha (a) bacterial streaming (b) and nematode
(c)
Morphological Characteristics
The identifications were based mainly on the basic morphological
characteristics with the aid of illustrations and descriptions from reference
materials such as books and journals.
Morphological
characteristics showed that the nematode are slender
especially the female. They are straight when relaxed. The basal knobs are
distinct. Vulva transverse and the lips are slightly protruding. The tail is
elongate-conoid ending in a simple blunt-spike. There is no sexual dimorphism
between the male and female. They are both essentially similar. The male tail is
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
Under La Trinidad, Benguet Condition / Agusta A. Allatiw. 2009
29
Plate 12. Nematodes seen under the binocular microscope: adult (a)
female tail (b) male tail (c)
arcuate when relaxed and has a simple blunt terminal spike. It has rosethorn-
shaped spicules (Plate 12).
Staining Reaction
Further identification through staining of plant tissues to make
nematodes showed different growth stages of the nematode such as stages such
as the eggs, juveniles, and adults within the tissues clearer under the microscope
(Plate 13). These were stained pink thus easily visualized under the microscope.
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
Under La Trinidad, Benguet Condition / Agusta A. Allatiw. 2009
30
a b
c d
Plate 13. Stained nematodes inside the leaf tissue eggs (a and b ) and
adults ( c and d)
Based on these initial diagnosis and identification, the nematode belongs
to the Family Aphelenchoididae specifically the genus Aphelenchoides, a foliar
nematode of important crops. Although majority of these species are free-living
and fungal feeders, a number of species have evolved to animal parasitism,
particularly on insects, and to plant parasitism. The nematodes A. besseyi, A.
fragariae and A. ritzemabozi are important pests of a number of crops worldwide
such as strawberry, chrysanthemum and rice. Typically, they feed on aerial parts
such as buds, leaves, flowers and fruits causing serious damages to their hosts
(Ferraz and Brown, 2002).
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
Under La Trinidad, Benguet Condition / Agusta A. Allatiw. 2009
31
Of all these important pests, the nematodes isolated and identified based
on their morphology coincide with the description of A. fragariae. In
strawberry, they cause the “strawberry crimp” disease. As reported by Hunt
(1999), it is also economically important on ornamental ferns, attacking 100
species which has an extensive host range mainly belonging to the Liliaceae,
Primulaceae, Pteridophyta and Ranunculaceae. A. fragariae is an ecto- and
endoparasite of the above-ground plant parts.
The female lays its eggs in the intercellular spaces of the leaves. The eggs
hatch and produce the four juvenile stage, and the adults all inside the leaf
(Agrios, 2005). They enter into the leaf via the stomata in damp conditions
(Klinger, 1970), although the epidermis can also be directly penetrated (Strϋmpel,
1967). The life cycle is completed in about 10-18 days at 18°C. They can survive as
adults in dead leaves or between scales of buds of infected tissues
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
Under La Trinidad, Benguet Condition / Agusta A. Allatiw. 2009
SUMMARY, CONCLUSION AND RECOMMENDATION
Summary
The study was conducted to characterize the pathogens associated with
the leaf disease complex of mondo grass. Sample leaves were taken from selected
sites in La Trinidad, Benguet and Baguio City.
The study was done by describing the symptoms manifested in diseased
leaves and signs gathered from the diseased fresh leaf specimens; isolating the
suspected pathogen into pure culture and describing them morphologically and
culturally in addition to microscopic observation and other confirmatory tests.
Results revealed that three organisms were associated with the mondo
grass disease complex. These are the fungus Fusarium sp., the bacterium
Xanthomonas sp. and the nematode Aphelenchoides fragariae. The symptoms
manifested by the leaves are either leaf spots, blights, yellowing or necrotic
lesions. The pathogens appear either singly or in complex. These may be
present singly or all at the same time in one symptom. However, these three
pathogens are most often observed to be present in same lesion/spot/blight.
The
Fusarium produces light pink mycelia with conidia with 4-6
septations which are similar to the conidia taken from the fresh infected leaves. It
likewise produces several microconidia.
The
bacterium
Xanthomonas is Gram negative. The growth on the
general and selected differential media is yellow. All species belonging to
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
Under La Trinidad, Benguet Condition / Agusta A. Allatiw. 2009
33
Xanthomonas is reported to be plant pathogens and are found only in association
with plants or plant materials. The Gram reaction, shape of the cells and the KOH
reaction indicate that the isolate is plant pathogenic.
The
nematode
Aphelenchoides is generally slender with distinct basal
knobs. Both male and female are similar having no sexual dimorphism. The tail
is elongate-conoid ending in blunt terminal spike. The nematode Aphelenchoides
fragariae is an important pest to economic crops such as the strawberry and other
ornamentals. Although the nematodes can cause diseases to plants alone, since
most of them are present in the soil, a disease interaction among other pathogens
living in the habitat is likely to occur.
Conclusions
From the results of the study, it is therefore concluded that there is an
interaction among the three pathogens resulting in a disease complex in mondo
grass.
The
fungus
Fusarium sp., the bacterium Xanthomonas sp. and the
nematode Aphelenchoides fragariae are pathogenic and these can pose a threat to
important crops. These pathogens can serve as a ready source of inoculum to
crops. In strawberry, the Aphelenchoides fragariae is a very important pathogen
causing short, bushy-looking plants with small distorted or wrinkled leaves.
Severe infection affects fruit buds leading to significant yield loss reduction
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
Under La Trinidad, Benguet Condition / Agusta A. Allatiw. 2009
34
Recommendations
With these findings, the following are recommended:
1. The mondo grass should not be used as ornamental crop in the
strawberry plantation areas.
2. The fungus Fusarium sp. should be identified further to the species
level. Its pathogenicity to other crops should be evaluated.
3. The bacterium Xanthomonas spp. should likewise be identified further
to the species level. Its pathogenicity to other crops should likewise be
evaluated.
4. Further survey of the distribution of the mondo grass in other places
should be done to assess the presence of these pathogens.
5. Uprooting or removal of mondo grass within the BSU campus is
suggested.
6. Pathogenicity test should be appropriately done especially on the
strawberry crop using the nematode Aphelenchoides fragariae for
further confirmation of its host range.
7. More studies must be done to determine which pathogen occurs first
during the interaction in relation to disease complex.
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
Under La Trinidad, Benguet Condition / Agusta A. Allatiw. 2009
LITERATURE CITED
AGRIOS, G.N. 2005. Plant Pathology. 5th edition. Elsevier Academic Press. Pp.
627.
____________. 1997. Plant Pathology. 4th edition. Elsevier Academic Press.
Pp. 803.
BENSON, H.J. 1998. Laboratory Manual in General Microbiology. 7th edition.
WBC/McGraw/Hill Companies, Inc. pp 46.
BILLING, E. 1987. Bacteria as plant pathogens. Wokingham: Van Nostrant
Reinhold.
BYRD, D. W. , T. KIRKPATRICK and K. R. BARKER. 1983. An improved
technique for clearing and staining plant tissues for detection of
nematodes. Journal of Nematology 15: 142-143.
CASTILLO, M. and T. REYES. 1972. Philippine Soil Nematodes: Inventory,
Classification and Key to their Identification. Philippine Phytopa-
thological Society. Pp. 57.
CIRCROLO, E.L., A. COLLMER, and A. GILLASPIE (eds.). 1987. Plant
pathogenic bacteria. Boston: Nijhoff.
DEPUTY, J. and D. HENSLEY. 1998. Ornamentals and Flowers. Cooperative
Extension Service. College of Tropical Agriculture and Human Resources
(CTAHR). University of Hawaii at Manoa. Posted at
www.ctahr.hawaii.edu/oc/freepubs/pdf/OF-28.pdf.
DICKSON, M. 2003. Molecular Plant Pathology. BIOS Scientific Publishers.
TJ International Ltd., Padstow, UK. Pp. 244.
FERRAZ, L. and D. BROWN. 2002. An Introduction to Nematodes: Plant
Nematology. A Student's Handbook. Pensoft Publishers. Acad. G.
Bonchev Str., B1. 6, 1113 Sofia, Bulgaria. Pp 221.
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
Under La Trinidad, Benguet Condition / Agusta A. Allatiw. 2009
36
GOSZCZYNSKA, T., J. J. SERFONTEIN and S. SERFONTEIN. 2000.
Introduction to Practical Bacteriology. A Manual for Phytobacteriology.
SAFRINET, The Southern African (SADC) LOOP of BIO-NET-
INTERNATIONAL. Bacterial Diseases Unit. ARC-Plant Protection
Research Institute. Pretonia South Africa. Pp 83.
HUNT, D. J. 1993. Aphelenchida, Longidoridae and Trichodoridae: Their
Systematics and Bionomics. CAB International. Wallingford Oxon
OX10 8DE, UK. Pp. 352.
KLINGLER, J. 1970. The reaction of Aphelenchoides fragariae to slit-like
micro-openings and to stomatal diffusion gases. Nematologica. Pp. 417-
422.
LOPEZ, R. H.; K. EVANS and J. BRIDGE. 2004. Plant Diseases Caused by
Nematodes. In: Nematology: Advances and Perspectives. Volume II:
Nematode Management and Utilization. Chen, Z. X.; S. Y. Chen, and
D. W. Dickson (eds.). CAB International. Pp. 1234.
LORENZEN, S. 1985. Phylogenetic aspects of pseudo-coelomate evolution.
The Origins and Relationships of Lower Invertebrates. Clarendon Press,
Oxford. Pp. 210-223.
LUCAS, J. A. 1998. Plant Pathology and Plant Pathogens. 3rd edition.
Blackwell Science Ltd. Pp. 274.
MANNERS, J. G. 1993. Principles of Plant Pathology 2nd edition. Cambridge
University Press. Pp. 27.
MOUNT, M.S. and G. H. LACEY. (eds.). 1993. Phytopathogenic prokaryotes,
Volume 2. Academic Press: New York.
MOORE, et. al. 1995. Botany. Wm. C. Brown Communications, Inc. pp. 599.
PERRY, R. N. and D. J. DWIGHT. 1998. The Physiology and Biochemistry
of Free-living and Plant Parasitic Nematodes. CABI Publishing. CAB
International, Wallingford. Oxon OX10 8DE, UK. Pp. 271.
PHILLIPS, R. and M. RIX. 1996. Late Perennials. Volume 2. Pan
Macmillan, Ltd. Pan Macmillan, 20 New Wharf Road, London. N1 9
RR. Basingstoke and Oxford.
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
Under La Trinidad, Benguet Condition / Agusta A. Allatiw. 2009
37
Plants Profile for Ophiopogon jaburan (lilyturf). United States Department of
Agriculture (USDA). Natural Resources Conservation Service. Posted at
http://plants.usda.gov/java/profile?symbol=OPJA3.
RIMANDO, T. J. 2001 Ornamental Horticulture: A Little Giant in the Tropics.
SEMEO Regional Center for Graduate Study and Research in Agriculture
(SEMEO SEARCA). UPLB, College, Laguna, Philippines. Pp. 333.
SCHAAD, N. W. and R. E. STALL. 1998. Laboratory Guide for
Identification of Plant Pathogenic Bacteria. 2nd edition. Bacteriology
Committee of the American Phytopathological Society. The American
Phytopathological Society. St. Paul Minnesota. Pp. 158.
STRÜMPEL, H. 1967. Beobachfungen zur Lebensweise von Aphelenchoides
fragariae in Lorraine-Begonien. Nematologica 13. pp. 67-72.
VIDAVER, A. K. 1967. Synthetic and complex media for rapid detection of
fluorescence of phytopathogenic pseudomonads: effects of carbon source.
Appl. Microbiol. 15:1523-1524.
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
Under La Trinidad, Benguet Condition / Agusta A. Allatiw. 2009
APPENDICES
APPENDIX TABLE 1. Composition and preparation of media
A. Nutrient Agar (NA)
Component Amount
(g/li)
Beef extract
3 g
Peptone 5
g
Agar 15
g
B. King’s Medium B Agar (KMBA)
Component
Amount (g/li)
MgSO4 (anhydrous)
1.5 g
K2HPO4 1.5
g
Proteose peptone
10 g
Agar 15
g
Glycerol 15
ml
The media was dispensed in individual 250 ml Erlenmeyer flask and
sterilized in an autoclave for 30 minutes at 15 pounds per square inch (psi).
C. Nutrient Glucose Agar (NGA)
Component Amount
(g/li)
Glucose 2.5
g
Agar 20
g
D. Sucrose Peptone Agar
Component Amount
(g/li)
Sucrose 20
g
K2HPO4 .5
g
MgSO4.7H2O .25
g
Agar 15
g
Adjust to pH 7.2
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
Under La Trinidad, Benguet Condition / Agusta A. Allatiw. 2009
39
E. Yeast Extract-Dextrose-CaCO3 Agar (YDCA)
Component Amount
(g/li)
Yeast 10
g
Dextrose 20
g
Calcium carbonate, USP light powder
20 g
Agar 15
g
All the components was dissolved in 1000 ml of distilled water except for
dextrose which was prepared separately into the flask with 100 ml distilled water.
The media was sterilized at 15 psi for 20 minutes. After sterilization, the dextrose
was mixed in a separate flask with the yeast, CaCO3 and agar. It was mixed
thoroughly by swirling the flask before pouring into the Petri plates. Observation
was done after 24 to 72 hours.
F. Potato Dextrose Agar
Component Amount
(g/li)
Potato 250
g
Dextrose 15
g
Agar 15
g
40
APPENDIX TABLE 2. Composition and preparation of reagents
A. Hucker’s ammonium oxalate crystal violet
Solution A
Crystal violet (90% dye content)
2.0 g
Ethyl
alcohol
(95%)
20.0
ml
Solution
B
Ammonium
oxalate
0.8
g
Distilled
water
80.0
ml
Mix solutions A and B. Filter through paper into storage bottle. Store for
24 hours before use.
B. Gram’s modification of Lugol’s solution
Iodine
5.0
g
Potassium
iodide
10.0
g
Distilled
water
100
ml
Mix potassium iodide with about 30 ml distilled water and dissolve. Add
iodine, bring to volume to 100 ml and dissolve the iodine several hours or
overnight in a dark place.
C. Decolorizing
1. Ethyl alcohol, 95%
2. Acetone: fastest agent
41
3. Acetone-alcohol: intermediate (95% ethyl alcohol, 100 ml; acetone,
100 ml)
With practice, any of the three decolorizing agents will yield good results.
D. Counterstaining
Stock
solution
Safranin
O
2.5
g
Ethyl
alcohol,
95%
100.0
ml
Working
solution
Stock
solution
10.0
ml
Distilled
water
90.0
ml
Staining procedure:
1. Make a smear by mixing a small amount of growth with a drop of
distilled water on a clean glass slide. Do not make thick smears for
they may not be decolorized as rapidly as the procedure requires.
2. Air dry and fix by passing slide filmside up quickly over the flame.
The slide should be hot but not burn the fingers. Do not scorch since
the cell wall may rupture causing Gram-positive organisms to accept
the counterstain.
3. Stain with ammonium oxalate crystal violet for 1 minute. Rinse
thoroughly but gently in tap water.
42
4. Cover smear with Gram iodine for 1 minute. Wash with tap water.
Shake off excess moisture.
5. Decolorize by dripping 95% ethyl alcohol over tilted slide for 10-15
seconds. Wash immediately with water.
6. Counterstain with safranin for 45 seconds. Wash with water.
7. Blot dry and examine under oil immersion objective.
Results: Gram-positive bacteria, purple to blue-black; Gram-negative
bacteria, red.
E. Gregersen’s method of determining gram stain reaction
1. Put 1-2 drops of 3% KOH on a glass slide.
2. Pick up a colony with a sterile loop and stir into KOH. After 5-10
seconds of stirring, raise the loop from the drop.
3. If KOH solution has become viscous and thread of slime follows the
loop, then organism is Gram-negative.
4. If there is no slime and a watery suspension remains, then organism is
Gram-positive.
F. Selective Staining Method for Nematode (Modified Technique by Byrd
et. al, 1983)
43
Carnoy’s solution
Component Amount
Absolute alcohol
60 ml
Chloroform 30
ml
Glacial acetic acid
10 ml
Staining solution ( Stock solution)
Component Amount
Acid fuchsin
3.5 g
Acetic acid
250 ml
Distilled water
750 ml
Pure glycerin (for preservation)
1. Wash all debris from the leaves, cut in 2 cm segments.
2. Place cut leaves in a beaker and cover with carnoy’s solution.
3. Leave in carnoy’s solution for a few days until the leaves are free
from chlorophyll.
5. When the leaves are free from chlorophyll, pour contents of beaker
onto sieve or mesh cloth and rinse thoroughly with running tap water.
6. Put leaves in 30 ml distilled water, add 1 ml stock solution for at least
a day for the stain to be absorbed by the nematodes. After staining,
rinse with distilled water and cover with pure glycerine.
RACHEL K. TUDAYAN. November 2006. Characterization of the Pathogens
Associated with the Disease Complex of Mondo Grass (Ophiopogon jaburan (Sieb.)
Lodd.). Benguet State University, La Trinidad, Benguet.
Adviser: Janet S. Luis, Ph.D.
ABSTRACT
The characterization of the pathogens infecting the mondo grass was done based
on symptoms, signs and cultural and morphological characteristics.
The disease symptoms observed includes leaf spots, blights, yellowing and
necrotic lesions. The pathogens appeared either singly or interacting together resulting in
a disease complex.
Three organisms were identified in the disease complex which include the fungus
Fusarium sp., the bacterium Xanthomonas sp. and the nematode Aphelenchoides
fragariae.
TABLE OF CONTENTS
Page
Bibliography …………………………………………………….
i
Abstract ………………………………………………………..
i
Table of Contents ……………………………………………...
ii
INTRODUCTON ……………………………………………..
1
REVIEW OF LITERATURE
The Host ……………………………………………………..
4
Disease ………………………………………………………
5
Diseases Caused by Bacteria ………………………………..
6
Diseases Caused by Fungi …………………………………..
8
Diseases Caused by Nematode ……………………………...
9
MATERIALS AND METHODS
Collection of Diseased Leaves ……………………………….
13
Description of Symptoms and Microscopic Observation
of Signs ……………………………………………………..
13
Identification of the Isolated Fungus ……………………...
13
Isolation …………………….…………………………...
13
Identification ……………………………………………
14
Identification of the Isolated Bacterium ……………………
14
Isolation ………………………………………………..
14
Identification …………………………………………..
15
Identification of the Foliar Nematode ……………………….
15
ii
Isolation ………………………………………………..
15
Identification …………………………………………..
15
Staining of plant tissues ……………………………...
16
Data Gathered ……………………………………………….
16
RESULTS AND DISCUSSIONS
Fungal Component of the Disease Complex ………………..
17
Disease symptoms and signs …………………………
17
Cultural characteristics ……………………………….
18
Morphological characteristics ………………………..
19
Bacterial Component of the Disease Complex ……………...
20
Disease symptoms and signs ………………………...
20
Cultural characteristics ……………………………….
22
Growth in differential media …………………………
23
Gram stain reaction ……………………………..........
24
Potassium hydroxide (KOH) test …………………….. 25
Nematode Component of the Disease Complex ……………. 26
Disease Symptoms and Signs …………………………... 26
Morphological Characteristics ………………………….. 28
Staining reaction ………………………………………... 29
SUMMARY, CONCLUSION, RECOMMENDATIONS ……. 32
LITERATURE CITED ………………………………………… 35
APPENDICES ………………………………………………... 38
iii
INTRODUCTION
Plants dominate the earth and are considered the most valuable things in
the natural world, affecting all aspects of human lives either directly or indirectly.
Human and animal lives depend mainly on the oxygen that these produce and
recycle and the ability to produce organic matter. Plants are the major source of
food and other basic necessities such as medicines, oils and fibers.
Plants contribute to the enhancement of the environment. These are used
in landscaping either as cover crops, shades and decorations. These aesthetic
purposes according to Rimando (2001), give warmth and color, peace and
serenity to the work area.
In the past centuries, humans learned to settle down and started cultivating
and domesticating plants and animals as sources of food supply. All over the
world, most of the fertile lands are already converted for agricultural use. In
reality, the food supply of human and animal population is determined by the
growth and productivity of plants.
Plant growth and productivity are, however, affected by several factors
one of which are diseases. The interaction between the plant and pathogenic
organism affects the food supply. Nowadays, a constant war occurs between the
farmer and these pathogens. The management of these diseases brings about
financial losses in terms of the use of chemical pesticides. These affect not only
the quantity but also the quality of the plant product causing considerable
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
Under La Trinidad, Benguet Condition / Agusta A. Allatiw. 2009
2
economic impact. On the other hand, symptoms of the disease in certain plants
may occur singly or in characteristic combinations. The causal organism may
also be due to one or more agents.
Mondo grass (Ophiopogon jaburan (Sieb.) Lodd.) which was introduced
in the Philippines during the early 1990’s (Madulid, ) is being used as a cover
crop or as a border plant in some of the localities within La Trinidad, Benguet and
Baguio City. Some are found to be planted along the roadside and or are
integrated with other ornamentals in a quadrangle.
It is observed, however, that most of these plants are attacked or infected
with disease. Symptoms of leaf spots, yellowing and browning of the green
leaves are observed.
It is therefore apparent to verify and identify the diseases of mondo grass
to determine if these pose a threat to other important crops such as vegetables,
fruits and ornamentals within the locality.
To determine the range of diseases attacking the plant and to fully
understand the nature and significance of these diseases, this study aimed to:
1. Describe the symptoms of the disease complex observed in mondo
grass;
2. Isolate and describe the pathogens associated with the disease
complex; and,
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
Under La Trinidad, Benguet Condition / Agusta A. Allatiw. 2009
3
3. Identify and characterize the pathogens attacking mondo grass based
on their cultural and morphological characteristics.
The study was conducted at the Plant Pathology Service Laboratory,
College of Agriculture, Benguet State University, La Trinidad, Benguet
from March to October 2006.
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
Under La Trinidad, Benguet Condition / Agusta A. Allatiw. 2009
4
REVIEW OF LITERATURE
Host
Ophiopogon or mondo grass is a perennial crop belonging to the
monocot group. It has the following plant classification
(http://plants.usda.gov.java/profile):
Kingdom …………………….Plantae – Plants
Subkingdom ………………..Tracheobionta – Vascular plants
Superdivision ……………..Spermatophyta – Seed plants
Division ………………….Magnaliophyta – Flowering plants
Class ……………………Liliopsida – Moncotyledons
Subclass ……………….Liliidae
Order
…………………Liliales
Family ……………….Liliaceae – Lily family
Genus ………………Ophiopogon Ker-Gawl
Species …………..Ophiopogon jaburan (Sieb.) Lodd.
Ophiopogon is native to the Honshu, Shikoku, and Kyushu,
provinces of Japan. It grows in woods and scrub at low altitudes,
flowering in July-August. This plant has thickened tuberous roots,
spreading by stolons to form loose patches. The leaves are narrow at 2-3
mm width commonly planted as ground cover or as low-maintenance
grass substitute (Phillip and Rix, 1996).
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
Under La Trinidad, Benguet Condition / Agusta A. Allatiw. 2009
5
Mondo grass is one closely related, grass-like groundcover. The
most frequently used species is Ophiopogon japonicus, which has several
popular varieties. These are generally drought-tolerant as well as disease-
and insect-resistant. Mondo grass is used for edging borders and in places
where minimal maintenance is required. Variegated selections can be
used as accent plants to add contrast and color
(www.ctahr.hawaii.edu/oc/freepubs/pdf/of.28.pdf).
The Disease
“Since it is not known whether plants feel pain or discomfort, as
plants do not speak or otherwise communicate to us, it is difficult to
pinpoint exactly when a plant is diseased” (Agrios, 1997).
To cause a disease, a pathogen must find a suitable host plant, pass
through the external protective layers of the host, and gain access to the
nutrients that it requires for its own growth and development (Dickson,
2003).
The emerging disease is dessiminated and spread by air, water,
insects and other vectors and most especially dessiminated by humans
through the tools, machineries and infected planting materials (Agrios,
2005). International trade also results in all sorts of plant materials being
moved from one place to place. Pathogens are often imported and become
established in countries where the conditions for disease development
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
Under La Trinidad, Benguet Condition / Agusta A. Allatiw. 2009
6
maybe more favorable than in the country of origin (Lucas, 1998). The
movement of plant and plant products is also an important source of new
diseases.
Diseases Caused by Bacteria
Bacteria are the most widely distributed, the simplest in
morphology, the smallest in size and the most difficult to identify. They
are prokaryotic and seldom photosynthetic (Benson, 1998).
There are a number of bacterial diseases of plants (Mount and
Lacy, 1982; Circrolo, Collmer and Gillaspie, 1987; Billing, 1987). Of the
1600 known bacterial species, 100 cause diseases in plants (Dickson,
2003). Most plant pathogenic bacteria are rod-shaped except
Streptomyces which is filamentous. Furthermore, most of the plant
pathogenic bacteria have delicate thread-like flagella, considerably longer
than the cells on which they are produced (Agrios, 2005).
Bacteria utilize a range of strategies for entry into plants. The leaf
surface can support large populations of many bacterial species that may
either be present in the air, splashed or carried by insects. These bacteria
can multiply on the surface to form microcolonies or larger aggregates
which enter into the plants through stomata, natural openings and wounds,
with the help of water droplets formed on the leaf surfaces. In some
Xanthomonas bacteria, they actively invade the plants through the
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
Under La Trinidad, Benguet Condition / Agusta A. Allatiw. 2009
7
hydathodes, the structure containing water pores located at leaf margins.
Under suitable conditions, copious amounts of fluid are exuded through
the hydathodes that are collected as guttation water drops around leaf
margins. The bacteria move chemotactically towards these drops, then
later is drawn back into the hydathodes carrying the bacteria with them in
the vascular system (Dickson, 2003.)
Bacteria can not enter plants via intact cuticles. Their entry is
through wounds and natural openings such as stomata or lenticels. The
presence of water is necessary to enable the pathogen to multiply and
move to a point where entry is possible. Inside the plant, they first
multiply in the intercellular spaces and/or the xylem and produce
pectolytic enzyme which causes cell death and the cells are then invaded
(Manners, 1993).
Agrios (2005) described the symptoms of bacteria such as leaf
spots and blights; soft rot of fruits, roots, and storage organs; wilts;
overgrowths; scabs and cankers. The most common type of disease in
plants are those that appear as spots of various sizes on leaves, stems,
blossoms and fruits. In some bacterial diseases, the spots continue to
advance rapidly and are then called blights.
In monocotyledonous plants, bacterial spots appear as streaks or
stripes. In humid weather conditions, infected tissues often exude masses
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
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8
of bacteria that spread to new tissues or plants and start new infections.
Almost all bacterial spots and blights of leaves, stems and fruits are caused
by bacteria belonging to the genera Pseudomonas and Xanthomonas.
Diseases Caused by Fungi
Fungi are usually filamentous, eukaryotic and spore-producing
organisms that lack chlorophyll. The cell walls are made up of chitin
combined with other complex carbohydrates and cellulose. All species of
fungi are either saprobes or symbionts. As symbionts, they may provide
certain benefits to their host, or may parasitize their host (Moore, et. al,
1995). The unit structure is the hypha, a branched filament consisting of a
row of cells with or without coenocytic structure. Reproduction usually
involves the production of unicellular or multicellular spores by sexual or
asexual processes.
Fungi may cause a very wide range of types of plant disease. More
than 10,000 species of fungi can cause disease in plants (Agrios, 2005). In
plant pathogenic fungi, there are sub-specific entities which attack
different host genera. All plant pathogenic fungi make use of extracellular
enzymes which they secrete at most stages of their attack on their hosts
(Manners, 1993).
Fungi and oomycetes are generally dispersed as spores that are
either deposited in the soil or transmitted through the air. Once the fungus
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9
adheres and germinates within a suitable host, it grows into the plant and
obtain its nutrients for its growth and development. It has the capacity to
do this either directly through the cuticle or grow towards the natural
opening or wound sites and enter through these (Dickson, 2003).
Fungi can cause local or general necrosis on plant tissues, and they
often cause reduced growth of plant organs or entire plants. Some of the
most common necrotic symptoms are leaf spot, blight, root-rot, damping
off, anthracnose, soft rot and dry rot. In many diseases, the fungal
pathogen grows or produces various structures on the surface of the host
which is of particular importance including mycelia, sclerotia,
sporophores, fruiting bodies and spores (Agrios, 2005).
Diseases Caused by Nematodes
Nematodes belong to the kingdom Animalia. Nematodes are
cylindrical worms, with a body shape commonly described as filiform or
having the shape of a thread. The name “Nematoda” is derived from the
Greek word nema, which means thread. They are aquatic animals that
inhabit oceans, seas, freshwater courses, body fluids, and in the film of
water present between soil particles and in plant which is of particular
importance.
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Plant parasitic nematodes are present in almost every type of
habitat with the limiting factors being moisture and food for survival.
These are obligate feeders on plants and can only obtain nutrients for
development and reproduction from the cytoplasm of living plant cells
(Perry and Wright, 1998). They constitute about 20% of the described
species (4,000 among 20,000) within the phylum Nematoda and are
included in the classes Chromadoria and Enoplea (Ferraz and Brown,
2002).
Nematodes are considered the most successful pseudo-coelomate
animals. This is due to their highly integrated body walls (cuticle,
epidermis and longitudinal muscles) that permit very efficient locomotion
(Lorenzen, 1985). Another contributing factor is the ability of some
nematodes to enter a state of anabiosis which permits long-term survival
under extremely harsh conditions. Nematodes have adapted to and are
capable of surviving a variety of extreme environmental/physical stresses
and even possess specialized developmental stages in order to do this
(Perry and Wright, 1998).
Female nematodes lay about 300-500 eggs. Depending on the
climate, the availability of hosts and the duration of each life cycle of the
particular nematode species may have from two or more than a dozen
generations per year.
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Symptoms caused by nematodes on roots may appear as root
lesions, root knots or root galls, excessive root and accompanied by plant
pathogenic or saprophytic bacteria and fungi as root rots. Certain species
of nematodes invade aboveground portions of plants. They cause galls,
necrotic lesions and rots, twisting or distortion of leaves and stems, and
abnormal development of the floral parts (Agrios, 2005).
Plant parasitic nematodes interact with many soil inhabitants
(fungi, micro-arthropods and free-living nematodes) and their soil
environment is significantly affected through human intervention with
agricultural procedures (Ferraz and Brown, 2002). Since most of the
nematodes live and operate in the soil where they are surrounded by many
soil inhabitants, there is a great interrelationship between the nematodes
and other plant pathogens. In many cases, an association develops
between nematodes and the other pathogens. Nematodes then become a
part of an etiological complex resulting in a combined pathogenic
potential that sometimes appears to be greater than the sum of damages
that either of the pathogens can produce individually (Agrios, 2005).
In the Philippines, published records show that nematodes are
prevalent in the Philippine soils. The plant parasitic forms apparently
have received much attention since they are frequent associates of plant
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12
diseases and are thus potential threat to agriculture (Castillo and Reyes,
1972).
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
Under La Trinidad, Benguet Condition / Agusta A. Allatiw. 2009
MATERIALS AND METHODS
A. Collection of Diseased Leaves
Sample leaves of infected mondo grass were collected, placed in
transparent polyethylene bags and brought to the laboratory for further diagnosis.
Samples were obtained from different locations in La Trinidad, Benguet and
Baguio City.
B. Description of Symptoms and Microscopic Observation of Signs
Symptoms of the diseased plants were described. To observe the signs,
thin sections and scrapings from infected fresh materials were mounted into a
drop of water on a glass slide then covered with cover slip and were studied under
the binocular compound microscope. All observations were recorded. Books,
journals and other reference materials on fungi, bacteria and nematodes were used
in characterizing the symptoms manifested by the mondo grass leaves and the
signs observed.
C. Identification of the Isolated Fungus.
Isolation. The specimens were washed with running tap water to get rid
of dirt, were cut into small sections about 2-5 sq. cm. then each piece of cut
tissues were disinfected with 10% sodium hypochlorite for 1-2 minutes. After
disinfection, the cut sections were rinsed with three changes of sterile distilled
water and were then blot-dried on a sterile tissue paper. Specimens were
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aseptically placed equidistantly on plated potato dextrose agar (PDA) then were
incubated for 3-5 days or more at 28-30°C. The culture medium was prepared
following the standard procedures. Pure culture of the fungal growth were re-
isolated in PDA slants, stored at 5°C which served as stock culture.
Identification. The cultural characteristics such as color and presence of
pigmentation were noted and recorded. The microorganisms were examined
under the electric binocular microscope taking note of their morphological
characteristics such as kind of mycelia produced, spore morphology and other
structures.
D. Identification of Isolated Bacterium
Isolation. The collected infected leaves were washed with tap water to
get rid of the surface dirt, disinfected with 10% sodium hypochlorite and were
finally rinsed with three changes of sterile distilled water. Leaf samples were
placed in a water blank and were macerated with a flamed glass rod to hasten the
oozing of bacterial cells. A loopful of the resulting solution was streaked onto
previously prepared nutrient agar (NA) which was prepared following the
standard procedures. The plates were incubated at 28-30°C for 24-72 hours. To
obtain a pure culture, a single colony of the bacterium was reisolated separately
into another plated NA. Stock cultures were kept on agar slants at 5°C.
Identification. To determine the genus of the isolated bacterium, the
following tests were done:
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1. Growth on various media
a. Yeast Extract Dextrose Agar (YDCA)
b. King’s Medium B Agar (KMBA)
c. Nutrient Glucose Agar (NGA)
d. Sucrose Peptone Agar (SPA)
2. Gram stain and potassium hydroxide (KOH) test
E. Identification of the Foliar Nematode
Isolation. Plant tissues were cut into longitudinal sections and were
placed in a glass Petri dish containing distilled water. The set/up was allowed to
stand for minutes to allow the nematodes to migrate and be released from the
damaged tissues. With the aid of a dissecting microscope, the nematodes were
then collected using an improvised hand picker (stick broom).
Identification. The mouth, stylet, tail and other distinguishing structures
of the isolated nematode were examined under the binocular electric microscope.
Identification was done with the aid of books, journals and other related available
references.
Staining of plant tissues. In order to observe the nematodes inside the
leaves, plant tissues were stained (modified technique) by cutting the leaves into
small sections about 2-3 sq. mm. then were soaked in a carnoy’s solution for a
few days until the chlorophyll pigmentation was removed. When the leaves
became clear, acid fuchsin (1 ml stock solution and 30 ml water) was added and
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was left for at least a day to be enable the stain to be absorbed by the nematode.
After staining, the leaves were rinsed in running tap water to remove excess stain
and were soaked and covered with pure glycerin.
Data Gathered
The following data were gathered:
1. Symptoms of the disease complex. These refer to the manifested
abnormalities on the diseased leaves of mondo grass.
2. Signs of pathogens involved in the disease complex. These refer to
the specific structures of the fungus, bacterium and nematode that
were freshly scraped or cut into thin sections from the diseased tissues
and observed under the binocular compound microscope.
3. Cultural and morphological characteristics of the isolates. These refer
to the color/pigmentation in PDA of the fungal isolate and gram stain,
KOH reaction and colony appearance in NA and other selected
differential media of the bacterial isolate.
4. Identification of pathogens involved in the disease complex. This was
limited to the genus level for the fungal and bacterial components of
the disease complex and species level for the nematode component.
5. Photodocumentation
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
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RESULTS AND DISCUSSION
Fungal Component of the Disease Complex
Disease Symptoms and Signs
Sample leaves exhibited circular to oval dark round spots on the leaves
surrounded by a definite yellow margin (Plate 1a). Other leaves showed blights
starting from the tip of the leaf moving downward (Plate 1b) . Other symptoms
observed showed association with other bacterial and nematode symptoms like
yellowing and necrotic spots.
a
b
Plate 1. Symptoms manifested by the suspected fungal component of the
disease complex appearing as leaf spot (a) and leaf blight (b)
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Plate 2. Signs observed under the compound microscope appearing as
septated boat-shaped numerous macroconidia with
microconidia
The microscopic examination of slide mounts prepared from thin sections
showed boat- or banana-shaped macroconidia with 4 to 6 septations. Numerous
microconidia were noted which imply the pathogenicity of the isolate (Plate 2).
Cultural Characteristics
The isolated fungus produces dense and compact light pink mycelia on
PDA. A pink-orange pigment is produced at the bottom of the plate but does not
diffuse throughout the medium (Plate 3).
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a b
Plate 3. Pure culture of Fusarium sp. with light pink mycelia at the top
(a) and pink-orange pigmentation at the bottom of the plate(b)
Morphological characteristics
Microscopic examination revealed slightly curved, banana-shaped
macroconidia with 4 to 6 septations. The septated mycelia are thick-walled.
There were numerous short and stout microconidia observed (Plate 4).
It should be noted that the type of macroconidia and microconidia
observed from the pure culture in PDA are exactly similar to those observed from
the fresh thin sections from diseased mondo grass leaves.
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Plate 4. Macroconidia and microconidia from the PDA culture of the
fungus
Bacterial Component of the Disease Complex
Disease Symptoms and Signs
Infected leaves manifested different kinds of symptoms appearing as leaf
spot and blight. Some symptoms have small dark brown lesions which start
from the leaf margin and moving longitudinally along the margin. Other
symptoms appear as spots of various sizes on the edge of the leaves then continue
to advance towards the healthy portion resulting in blights. Some symptoms
first appeared at the tip of the leaves then moving downward (Plate 5a). During
the rainy season, the symptoms had a yellowish and water-soaked appearance
(Plate 5b).
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a
b
Plate 5. Symptoms manifested by the suspected bacterial component of
the disease complex appearing as small dark brown lesions (a)
and yellowish water-soaked appearance (b)
Plate 6. Bacterial streaming from cut tissues
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The microscopic observation of the thin sections prepared from the
diseased leaves showed massive bacterial streaming especially those taken from
the lesions with dark brown discoloration (Plate 6). Bacterial streaming is one
property that is synonymous to phytopathogenic bacteria which are motile due to
the presence of flagella. Motility can be readily observed especially if the
material is fresh (Goszczynska, et. al, 2000).
Cultural characteristics
In NA, 72-hour old cultures of the suspected bacterium had yellow
colonies (Plate 7). The yellow color looked like an egg white with egg yolk at the
middle when seen through light which is a typical indication that the organism
involved belongs to the genus Xanthomonas (Lando, personal communication).
Plate 7. Bacterial isolate in NA 72 hours after isolation
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a b
c d
Plate 8. Bacterial isolate in differential media appearing as dark
yellow in YDCA (a) and light yellow in KMBA (b) SPA
( c) and NGA (d)
Confirmatory identification in differential media revealed dark yellow
colonies on YDCA and light yellow color on the other media (Plate 8). These
characteristics confirm the description of Schaad and Stall (1998). The colonies
are mucoid, convex and shiny. The yellow membrane-bound pigments are
brominated arylpolyne esters (xanthomonadins).
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Gram Stain Reaction
Under the oil immersion objective (OIO), the stained bacteria are rod-
shaped and appear pink in color which indicates that these absorb the color of the
counterstain (safranin) thereby are classified as Gram-negative (Plate 9a.) Most
phytopathogenic bacteria are Gram-negative and rod-shaped (Goszczynska, et. al,
2000). Bacteria that cause leaf spot or blight are often Gram-negative and may
belong to Pseudomonas or Xanthomonas (Agrios, 2005).
a b
Plate 9. Rod-shaped Gram-negative bacterial cells from smear prepared
from the NA culture (a) and mucoid thread produced from the
KOH test done on slide suspension of the suspected bacterial
isolate (b)
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Potassium Hydroxide Solubility (KOH) Test
The preliminary diagnostic identification of a bacterium using
potassium hydroxide (KOH - 3% aq., w/v) test showed a mucoid thread produced
indicating that the bacterium is Gram-negative (Plate 9b).
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Nematode Component of the Disease Complex
Disease Symptoms and Signs
The above-ground symptoms observed vary especially on the leaves.
These appear as necrotic lesions then advance towards the healthy leaves where
lesions coalesce. The affected tissues turn brown and sometimes discolored. Just
above the ground, the leaves appear yellowish turning brown with time causing
an early senescence of the leaves (Fig 10). Other symptoms manifested are leaf
blotching and yellowing while others are similar to the symptoms manifested by
the fungus and bacterium.
These observations indicate the presence of the three pathogens in the
disease complex. The sample plants collected showed that most often, these
organisms are all present at a time when examined under the microscope (Fig 11).
Frequently, nematodes facilitate the entry and establishment of other plant
pathogens. They may either have an “interrelationship” and “interaction” with
bacteria, fungi and viruses (Lopez et. al., 2004).
In 1892, interactions of nematode with other plant pathogens had already
been recorded. All plant pathogenic nematodes cause wound in plants by
puncturing, rupturing or by separating cells thereby introducing or aiding the
entry of other pathogens already present on the plant surface. Chen et. al. (2004)
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Plate 10. Symptoms manifested by suspected nematode component
of the disease complex appearing as yellow to brown
discoloration starting at the base of the leaves.
cited some reports on nematode-fungi interactions like: nematode with Fusarium
oxysporum and other Fusarium spp., Verticillium spp., Pythium spp., and
Cylindrocarpon spp. Bacteria-nematode interactions are also known to occur:
like nematode with Clavibacter spp., Pseudomonas spp. and Agrobacterium spp.
For viruses, the nematode vector belongs to the genera Xiphinema, Trichodorus
and Longidorus.
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a
c
b
Plate 11. Disease complex in the mondo grass as indicated by the
presence of hypha (a) bacterial streaming (b) and nematode
(c)
Morphological Characteristics
The identifications were based mainly on the basic morphological
characteristics with the aid of illustrations and descriptions from reference
materials such as books and journals.
Morphological
characteristics showed that the nematode are slender
especially the female. They are straight when relaxed. The basal knobs are
distinct. Vulva transverse and the lips are slightly protruding. The tail is
elongate-conoid ending in a simple blunt-spike. There is no sexual dimorphism
between the male and female. They are both essentially similar. The male tail is
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Plate 12. Nematodes seen under the binocular microscope: adult (a)
female tail (b) male tail (c)
arcuate when relaxed and has a simple blunt terminal spike. It has rosethorn-
shaped spicules (Plate 12).
Staining Reaction
Further identification through staining of plant tissues to make
nematodes showed different growth stages of the nematode such as stages such
as the eggs, juveniles, and adults within the tissues clearer under the microscope
(Plate 13). These were stained pink thus easily visualized under the microscope.
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a b
c d
Plate 13. Stained nematodes inside the leaf tissue eggs (a and b ) and
adults ( c and d)
Based on these initial diagnosis and identification, the nematode belongs
to the Family Aphelenchoididae specifically the genus Aphelenchoides, a foliar
nematode of important crops. Although majority of these species are free-living
and fungal feeders, a number of species have evolved to animal parasitism,
particularly on insects, and to plant parasitism. The nematodes A. besseyi, A.
fragariae and A. ritzemabozi are important pests of a number of crops worldwide
such as strawberry, chrysanthemum and rice. Typically, they feed on aerial parts
such as buds, leaves, flowers and fruits causing serious damages to their hosts
(Ferraz and Brown, 2002).
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Of all these important pests, the nematodes isolated and identified based
on their morphology coincide with the description of A. fragariae. In
strawberry, they cause the “strawberry crimp” disease. As reported by Hunt
(1999), it is also economically important on ornamental ferns, attacking 100
species which has an extensive host range mainly belonging to the Liliaceae,
Primulaceae, Pteridophyta and Ranunculaceae. A. fragariae is an ecto- and
endoparasite of the above-ground plant parts.
The female lays its eggs in the intercellular spaces of the leaves. The eggs
hatch and produce the four juvenile stage, and the adults all inside the leaf
(Agrios, 2005). They enter into the leaf via the stomata in damp conditions
(Klinger, 1970), although the epidermis can also be directly penetrated (Strϋmpel,
1967). The life cycle is completed in about 10-18 days at 18°C. They can survive as
adults in dead leaves or between scales of buds of infected tissues
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
Under La Trinidad, Benguet Condition / Agusta A. Allatiw. 2009
SUMMARY, CONCLUSION AND RECOMMENDATION
Summary
The study was conducted to characterize the pathogens associated with
the leaf disease complex of mondo grass. Sample leaves were taken from selected
sites in La Trinidad, Benguet and Baguio City.
The study was done by describing the symptoms manifested in diseased
leaves and signs gathered from the diseased fresh leaf specimens; isolating the
suspected pathogen into pure culture and describing them morphologically and
culturally in addition to microscopic observation and other confirmatory tests.
Results revealed that three organisms were associated with the mondo
grass disease complex. These are the fungus Fusarium sp., the bacterium
Xanthomonas sp. and the nematode Aphelenchoides fragariae. The symptoms
manifested by the leaves are either leaf spots, blights, yellowing or necrotic
lesions. The pathogens appear either singly or in complex. These may be
present singly or all at the same time in one symptom. However, these three
pathogens are most often observed to be present in same lesion/spot/blight.
The
Fusarium produces light pink mycelia with conidia with 4-6
septations which are similar to the conidia taken from the fresh infected leaves. It
likewise produces several microconidia.
The
bacterium
Xanthomonas is Gram negative. The growth on the
general and selected differential media is yellow. All species belonging to
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
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33
Xanthomonas is reported to be plant pathogens and are found only in association
with plants or plant materials. The Gram reaction, shape of the cells and the KOH
reaction indicate that the isolate is plant pathogenic.
The
nematode
Aphelenchoides is generally slender with distinct basal
knobs. Both male and female are similar having no sexual dimorphism. The tail
is elongate-conoid ending in blunt terminal spike. The nematode Aphelenchoides
fragariae is an important pest to economic crops such as the strawberry and other
ornamentals. Although the nematodes can cause diseases to plants alone, since
most of them are present in the soil, a disease interaction among other pathogens
living in the habitat is likely to occur.
Conclusions
From the results of the study, it is therefore concluded that there is an
interaction among the three pathogens resulting in a disease complex in mondo
grass.
The
fungus
Fusarium sp., the bacterium Xanthomonas sp. and the
nematode Aphelenchoides fragariae are pathogenic and these can pose a threat to
important crops. These pathogens can serve as a ready source of inoculum to
crops. In strawberry, the Aphelenchoides fragariae is a very important pathogen
causing short, bushy-looking plants with small distorted or wrinkled leaves.
Severe infection affects fruit buds leading to significant yield loss reduction
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
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34
Recommendations
With these findings, the following are recommended:
1. The mondo grass should not be used as ornamental crop in the
strawberry plantation areas.
2. The fungus Fusarium sp. should be identified further to the species
level. Its pathogenicity to other crops should be evaluated.
3. The bacterium Xanthomonas spp. should likewise be identified further
to the species level. Its pathogenicity to other crops should likewise be
evaluated.
4. Further survey of the distribution of the mondo grass in other places
should be done to assess the presence of these pathogens.
5. Uprooting or removal of mondo grass within the BSU campus is
suggested.
6. Pathogenicity test should be appropriately done especially on the
strawberry crop using the nematode Aphelenchoides fragariae for
further confirmation of its host range.
7. More studies must be done to determine which pathogen occurs first
during the interaction in relation to disease complex.
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Macmillan, Ltd. Pan Macmillan, 20 New Wharf Road, London. N1 9
RR. Basingstoke and Oxford.
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
Under La Trinidad, Benguet Condition / Agusta A. Allatiw. 2009
37
Plants Profile for Ophiopogon jaburan (lilyturf). United States Department of
Agriculture (USDA). Natural Resources Conservation Service. Posted at
http://plants.usda.gov/java/profile?symbol=OPJA3.
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SEMEO Regional Center for Graduate Study and Research in Agriculture
(SEMEO SEARCA). UPLB, College, Laguna, Philippines. Pp. 333.
SCHAAD, N. W. and R. E. STALL. 1998. Laboratory Guide for
Identification of Plant Pathogenic Bacteria. 2nd edition. Bacteriology
Committee of the American Phytopathological Society. The American
Phytopathological Society. St. Paul Minnesota. Pp. 158.
STRÜMPEL, H. 1967. Beobachfungen zur Lebensweise von Aphelenchoides
fragariae in Lorraine-Begonien. Nematologica 13. pp. 67-72.
VIDAVER, A. K. 1967. Synthetic and complex media for rapid detection of
fluorescence of phytopathogenic pseudomonads: effects of carbon source.
Appl. Microbiol. 15:1523-1524.
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
Under La Trinidad, Benguet Condition / Agusta A. Allatiw. 2009
APPENDICES
APPENDIX TABLE 1. Composition and preparation of media
A. Nutrient Agar (NA)
Component Amount
(g/li)
Beef extract
3 g
Peptone 5
g
Agar 15
g
B. King’s Medium B Agar (KMBA)
Component
Amount (g/li)
MgSO4 (anhydrous)
1.5 g
K2HPO4 1.5
g
Proteose peptone
10 g
Agar 15
g
Glycerol 15
ml
The media was dispensed in individual 250 ml Erlenmeyer flask and
sterilized in an autoclave for 30 minutes at 15 pounds per square inch (psi).
C. Nutrient Glucose Agar (NGA)
Component Amount
(g/li)
Glucose 2.5
g
Agar 20
g
D. Sucrose Peptone Agar
Component Amount
(g/li)
Sucrose 20
g
K2HPO4 .5
g
MgSO4.7H2O .25
g
Agar 15
g
Adjust to pH 7.2
Aboveground Biomass Production of Different Agroforestry Hedgerow Species
Under La Trinidad, Benguet Condition / Agusta A. Allatiw. 2009
39
E. Yeast Extract-Dextrose-CaCO3 Agar (YDCA)
Component Amount
(g/li)
Yeast 10
g
Dextrose 20
g
Calcium carbonate, USP light powder
20 g
Agar 15
g
All the components was dissolved in 1000 ml of distilled water except for
dextrose which was prepared separately into the flask with 100 ml distilled water.
The media was sterilized at 15 psi for 20 minutes. After sterilization, the dextrose
was mixed in a separate flask with the yeast, CaCO3 and agar. It was mixed
thoroughly by swirling the flask before pouring into the Petri plates. Observation
was done after 24 to 72 hours.
F. Potato Dextrose Agar
Component Amount
(g/li)
Potato 250
g
Dextrose 15
g
Agar 15
g
40
APPENDIX TABLE 2. Composition and preparation of reagents
A. Hucker’s ammonium oxalate crystal violet
Solution A
Crystal violet (90% dye content)
2.0 g
Ethyl
alcohol
(95%)
20.0
ml
Solution
B
Ammonium
oxalate
0.8
g
Distilled
water
80.0
ml
Mix solutions A and B. Filter through paper into storage bottle. Store for
24 hours before use.
B. Gram’s modification of Lugol’s solution
Iodine
5.0
g
Potassium
iodide
10.0
g
Distilled
water
100
ml
Mix potassium iodide with about 30 ml distilled water and dissolve. Add
iodine, bring to volume to 100 ml and dissolve the iodine several hours or
overnight in a dark place.
C. Decolorizing
1. Ethyl alcohol, 95%
2. Acetone: fastest agent
41
3. Acetone-alcohol: intermediate (95% ethyl alcohol, 100 ml; acetone,
100 ml)
With practice, any of the three decolorizing agents will yield good results.
D. Counterstaining
Stock
solution
Safranin
O
2.5
g
Ethyl
alcohol,
95%
100.0
ml
Working
solution
Stock
solution
10.0
ml
Distilled
water
90.0
ml
Staining procedure:
1. Make a smear by mixing a small amount of growth with a drop of
distilled water on a clean glass slide. Do not make thick smears for
they may not be decolorized as rapidly as the procedure requires.
2. Air dry and fix by passing slide filmside up quickly over the flame.
The slide should be hot but not burn the fingers. Do not scorch since
the cell wall may rupture causing Gram-positive organisms to accept
the counterstain.
3. Stain with ammonium oxalate crystal violet for 1 minute. Rinse
thoroughly but gently in tap water.
42
4. Cover smear with Gram iodine for 1 minute. Wash with tap water.
Shake off excess moisture.
5. Decolorize by dripping 95% ethyl alcohol over tilted slide for 10-15
seconds. Wash immediately with water.
6. Counterstain with safranin for 45 seconds. Wash with water.
7. Blot dry and examine under oil immersion objective.
Results: Gram-positive bacteria, purple to blue-black; Gram-negative
bacteria, red.
E. Gregersen’s method of determining gram stain reaction
1. Put 1-2 drops of 3% KOH on a glass slide.
2. Pick up a colony with a sterile loop and stir into KOH. After 5-10
seconds of stirring, raise the loop from the drop.
3. If KOH solution has become viscous and thread of slime follows the
loop, then organism is Gram-negative.
4. If there is no slime and a watery suspension remains, then organism is
Gram-positive.
F. Selective Staining Method for Nematode (Modified Technique by Byrd
et. al, 1983)
43
Carnoy’s solution
Component Amount
Absolute alcohol
60 ml
Chloroform 30
ml
Glacial acetic acid
10 ml
Staining solution ( Stock solution)
Component Amount
Acid fuchsin
3.5 g
Acetic acid
250 ml
Distilled water
750 ml
Pure glycerin (for preservation)
1. Wash all debris from the leaves, cut in 2 cm segments.
2. Place cut leaves in a beaker and cover with carnoy’s solution.
3. Leave in carnoy’s solution for a few days until the leaves are free
from chlorophyll.
5. When the leaves are free from chlorophyll, pour contents of beaker
onto sieve or mesh cloth and rinse thoroughly with running tap water.
6. Put leaves in 30 ml distilled water, add 1 ml stock solution for at least
a day for the stain to be absorbed by the nematodes. After staining,
rinse with distilled water and cover with pure glycerine.
Document Outline
- Characterization of the Pathogens
Associated with the Disease Complex of Mondo Grass (Ophiopogon jaburan (Sieb.)
Lodd.).
- BIBLIOGRAPHY
- ABSTRACT
- TABLE OF CONTENTS
- INTRODUCTION
- REVIEW OF LITERATURE
- Host
- The Disease
- Diseases Caused by Bacteria
- Diseases Caused by Fungi
- Diseases Caused by Nematodes
- MATERIALS AND METHODS
- Collection of Diseased Leaves
- Description of Symptoms and Microscopic Observation of Signs
- Identification of the Isolated Fungus.
- Identification of Isolated Bacterium
- Identification of the Foliar Nematode
- Data Gathered
- RESULTS AND DISCUSSION
- Disease Symptoms and Signs
- Cultural Characteristics
- Morphological characteristics
- Disease Symptoms and Signs
- Cultural characteristics
- Gram Stain Reaction
- Potassium Hydroxide Solubility (KOH) Test
- SUMMARY, CONCLUSION AND RECOMMENDATION
- Summary
- Conclusions
- Recommendations
- LITERATURE CITED
- APPENDICES