Screening the antagonistic ability to pathogenic microorganisms and researching biochemical characteristics of streptomycetes sp vnua23

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE
FACULTY OF BIOTECHNOLOGY
--------------

GRADUATION THESIS
TITTLE:
“SCREENING THE ANTAGONISTIC ABILITY TO PATHOGENIC
MICROORGANISMS AND RESEARCHING BIOCHEMICAL
CHARACTERISTICS OF STREPTOMYCETES SP. VNUA23”

Hanoi – 2022


VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE
FACULTY OF BIOTECHNOLOGY
--------------

GRADUATION THESIS
TITTLE:

“SCREENING THE ANTAGONISTIC ABILITY TO
PATHOGENIC MICROORGANISMS AND
RESEARCHING BIOCHEMICAL CHARACTERISTICS
OF STREPTOMYCETES SP. VNUA23”

Practicing student’s name

: NGUYEN BINH NAM

Class

: K62-CNSHE

Student’s code

: 620443

Supervisor

: Dr. TRỊNH XUÂN HOẠT
MSc. TRẦN THỊ HỒNG HẠNH

Major

: Microbial Technology

Hanoi – 2022
ii


COMMITMENT
I hereby declare that the data and research results in this thesis are true
and do not copy the results of any previous graduate reports.
Graduation thesis with references to documents, citation information is
indicated in the references section.
Hanoi, March 15th, 2022
Sincerely

Nguyen Binh Nam

iii

ACKNOWLEDGEMENTS
Firstly, I would like to express my special thanks of gratitude to my
teacher who gave me the golden opportunity to do this wonderful project on the
topic, which also helped me in doing a lot of research and I came to know about
so many new things I am really thankful to him.
Secondly, I would also like to thank my parents and best friends who
helped me a lot in finalizing this project within the limited time frame. My
completion of this project could not have been accomplished without the support
of them.
At last but not least, I am thankful to all my teachers and friends who have
been always helping and encouraging me though out the year. I have no valuable
words to express my thanks, but my heart is still full of the favours received
from every person.

Hanoi, March 15th, 2022
Sincerely

Nguyen Binh Nam

iv


INDEX

COMMITMENT ............................................................................................................ ii
ACKNOWLEDGEMENTS ...........................................................................................iv
INDEX ............................................................................................................................. v
LIST OF TABLES ....................................................................................................... vii

LIST OF FIGURES ..................................................................................................... viii
LIST OF ABBREVIATIONS ........................................................................................ix
ABSTRACT ....................................................................................................................x
PART I. INTRODUCTION ............................................................................................ 1
I. Introduction ..................................................................................................................1
1.1. Problem .....................................................................................................................1
1.2. Research purpose ......................................................................................................2
PART II. LITERATURE REVIEW ................................................................................3
2.1. Overview of plant pathogens ....................................................................................3
2.1.1. Overview of plant pathogenic fungi ......................................................................3
2.1.2. Over view of plant pathogenic bacteria ............................................................... 10
2.2. Overview of Actinobacteria ...................................................................................18
2.2.1. General introduction to Actinomycetes ................................................................ 18
2.2.2. Overview of Streptomyces ...................................................................................20
PART III. MATERIALS AND METHODS .................................................................28
3.1. Materials .................................................................................................................28
3.1.1. Location and time of the study ............................................................................28
3.1.2. Materials ..............................................................................................................28
3.1.3. Equipments ..........................................................................................................28
3.1.4. Medium................................................................................................................29
3.2. Methods ..................................................................................................................29
3.2.1. Screening the antagonistic ability of Streptomyces sp. VNUA23 to pathogenic
organisms .......................................................................................................................29

v


3.2.2. Biochemical characteristics of Streptomyces sp. VNUA 23 ............................... 30
CHAPTER IV. RESULTS AND DISCUSSION .......................................................... 40
4.1. Screening and the antagonistic ability of Streptomyces sp. VNUA 23 ..................40

4.1.1. Antifungal ............................................................................................................40
4.1.2. Antibacterial ........................................................................................................41
4.2. Biochemical properties of Streptomyces sp. VNUA23 ..........................................42
4.2.1. Ability to produce extracellular enzymes ............................................................ 42
4.2.2. Ability to produce IAA ........................................................................................43
4.2.3 Ability to produce hydro sulfide...........................................................................45
4.2.4. Ability to produce indole .....................................................................................46
4.2.5. Methyl red test .....................................................................................................47
4.2.6. Voges–Proskauer test .......................................................................................... 48
4.2.7. Ability to utilize citrate ........................................................................................49
4.2.8. Ability to hydrolize gelatin ..................................................................................49
4.2.9. Ability to solubilize phosphate ............................................................................50
4.2.10. Ability to produce urease...................................................................................51
4.2.11. Ability to produce siderophore ..........................................................................52
4.2.12. Ability to reduce nitrate .....................................................................................53
CHAPTER V. CONCLUSION AND PROPOSAL ......................................................54
5.1. Conclusion ..............................................................................................................55
5.2. Proposal ..................................................................................................................55
REFFERENCES ............................................................................................................56

vi


LIST OF TABLES
Table 2.1. The ability to use different carbon sources of actinomycetes
VNUA23 ............................................................................................. 25
Table 3.1 Ingredients for medium ....................................................................... 31
Table 3.2 Standard IAA Solution Composition .................................................. 32
Table 3.3 Ingrediants for Indole test ................................................................... 34
Table 3. 4. Gelatin hydrolysis medium ............................................................... 36

vii


LIST OF FIGURES
Figure 2.1 Morphological characteristics of VNUA23 ....................................... 23
Figure 2.2. Morphology of the fiber (A), Spore and spore production (B and C)
of VNUA23 ......................................................................................................... 23
Figure 2.3 Inoculation of VNUA23 on ISP6 medium after 7 days of culture .... 24
Figure 2.4 The ability to grow of VNUA23 at 20, 30, 37, 40, 45 and 50℃........ 24
Figure 4.1 Antifungal activity of VNUA23 against C. Gloeosporioides (55.56%) 40
Figure 4.2 Antifungal activity of VNUA23 agaisnt F.solani (HT39) (37.50%) 40
Figure 4.3 Antibacterial activity of VNUA23 against Xanthomonas axonopodis(a);
Clavibacter michiganesis(b) and Ralstonia solanacearum(c) ............................ 41
Figure 4.4 Results of six differences enzyme activity: (a) Catalase, (b) Protease,
(c) pectinase, (d) Amylase, (e) Cellulase, (f) Chitinase ...................................... 43
Figure 4.5 IAA standard curve ............................................................................ 43
Figure 4.6 Result of IAA test .............................................................................. 44
Figure 4.7 Result of sulphur reduction test ......................................................... 46
Figure 4.8 Result of Indole test ........................................................................... 46
Figure 4.9 Result of MR test ............................................................................... 47
Figure 4.10 Result of VP test .............................................................................. 48
Figure 4.11 Result of citrate test ......................................................................... 49
Figure 4.12 Result of gelatinase test ................................................................... 50
Figure 4.13 Result of phosphorus solubilizing ability ........................................ 51
Figure 4.14 Result of Urearase test ..................................................................... 51
Figure 4.15 Result of Siderophore test................................................................ 53
Figure 4.16 Result of Nitrate test ........................................................................ 54

viii

LIST OF ABBREVIATIONS
Abbreviation

Full word

Approx.

Approximately

Spp

Several species

FSSC

Fusarium solani species complex

F.solani

Fusarium solani

Sp.

specie

SDS

sudden death syndrome

C. gloeosporioides

colletotrichum gloeosporioides

C. higginsianum

Colletotrichum higginsianum

C. acutatum

Colletotrichum acutatum

pv.

Pathovar

R. solanacearum

ralstonia solanacearum

Cmm

Clavibacter michiganensis sp. michiganensis

ix


ABSTRACT
For the purpose of the study, to investigate the antagonism ability of

VNUA23 with some pathogenic bacteria of plants and animals for application in
bio-fertilizer production, I conducted a survey on the antagonism ability of
VNUA23 with bacteria causing disease and pathogenic bacteria Xanthomonas
axonopodis, Rastonia solanacearum, Clavibacter michiganensis and also
surveying other biochemical characteristics such as production of Siderophore,
IAA, ..., ability to degrade insoluble phosphate or produce extracellular enzymes
that can be applied in the production of microbial fertilizers of VNUA23.
From the material source is Streptomyces sp. VNUA23 at the Vietnam
National University of Agriculture, I activated and stored VNUA23 on Gause I
and ISP2 medium to do survey experiments. Streptomyces sp. VNUA23 has
almost

no

antagonism

against

Xanthomonas

axonopodis,

Rastonia

solanacearum, Clavibacter michiganensis but has the ability to antagonize
Fusarium solani and Collectotrichum gloeosporioides.
In addition, VNUA23 also has the ability to degrade insoluble phosphate,
produce siderophore and many other enzymes.

x

PART I. INTRODUCTION
I. Introduction
1.1. Problem
Crop diseases not only cause significant crop damage in Vietnam but also
in other parts of Southeast Asia and around the world. Outbreaks of diseases in
economically important crops can have a major impact on individual farmers in
localities where there are few suitable substitutes. (Kannan et al., 2015)
reported, approx. 7100 species of plant pathogenic organisms including viruses,
bacteria, fungi, nematodes and insects, of which about 150 species of bacteria
cause plant diseases. Bacterial diseases are common in tropical regions. Many
diseases are caused by bacteria, including bacterial wilt, leaf spot, leaf burn,
swelling and ulceration. Some species also cause rot in fruits and vegetables
before and after harvest. In general, plant pathogens of the Xanthomonadaceae,
Pseudomonadaceae and Enterobacteriaceae families attack all types of plants
that can provide suitable food and shelter. The most damaging plant pathogens
belong to genera such as Erwinia, Pectobacterium, Pantoea, Agrobacterium,
Pseudomonas, Ralstonia, Burkholderia, Acidovorax, Xanthomonas, Clavibacter,
Xylella, Spiroplasma and Phytoplasma.
Fungi have also developed a variety of techniques for colonizing plants,
and these interactions result in a wide range of consequences, from beneficial
interactions to host death. In terms of plant infections, fungi are perhaps the
most varied category of ecologically and economically significant hazards.
Fungal plant pathogens are mostly found in the phyla Ascomycota and
Basidiomycota.
Cladosporium

Plant
spp.),

diseases

classified

Sordariomycetes

as

(e.g.,

Dothideomycetes
Magnaporthe

spp.),

(e.g.,
or

Leotiomycetes (e.g., Magnaporthe spp). (e.g., Botrytis spp.). Basidiomycetes
include the two biggest plant disease groups: rusts (Pucciniomycetes) and smuts
1


(spread among the subphylum of Ustilaginomycotina)(Doehlemann et al.,
2017a).
The general trend of the world is towards a sustainable agriculture,
therefore, using microbial strains in biological disease prevention and treatment
is the most preferred because of its environmental friendliness, without affecting
the product quality. Among those groups of biological agents, actinomycetes is a

group of microorganisms that have been studied a lot because of their great
potential in biological fight and control of plant diseases, secrete antibiotics.
Besides, actinomycetes can also stimulate disease resistance as well as help
plants to be resistant to adverse environmental conditions (Hasegawa et al.,
2006).
Study of actinomycetes in the management of plant diseases are very
necessary and urgent, so I made this thesis with the name: SCREENING THE
ANTAGONISTIC ABILITY TO PATHOGENIC MICROORGANISMS
AND

RESEARCHING

BIOCHEMICAL

CHARACTERISTICS

OF

STREPTOMYCETES sp. VNUA23.
1.2. Research purpose
 Investigation of the antagonistic activity of VNUA23 against some plant
diseases.
 Investigation of some biochemical characteristics of VNUA23.
 Screening and selecting potential antagonistic actinomycetes.
 Evaluation of the basic antagonistic effects of actinomycetes against
pathogenic fungi and bacteria.

2

PART II. LITERATURE REVIEW
2.1. Overview of plant pathogens
2.1.1. Overview of plant pathogenic fungi
Phytopathogenic fungi have been catastrophic hazards to agriculture
throughout history. Plant illnesses were blamed on gods in ancient times, which
was a primary driving force in the creation of religious beliefs. Demeter was
worshiped as the goddess of grain and fertility in Greece from around 1300 BC.
Ceres appears as Demeter's feminine attachment in the ancient Roman empire
beginning in the fifth century BC. The deity Robigus, who was the personal
expression of agricultural sickness, was honored with a spring festival
(Robigalia) in ancient Roman religion. This illness (Robigo) is a kind of wheat
rust that causes a red tint, and it was mythologically associated with Mars, a god
of both agriculture and violence. To appease Robigus and save crops from being
devastated by rust disease, the Romans sacrificed animals - probably with no
effectiveness. Theophrastus (370 – 288 BC), an Aristotelian student, made the
first meaningful scientific contribution to the understanding of fungal plant
disease. He recorded the occurrence of rust infections on various host plants and
in the setting of the landscape in his botanical research.
The fungus can be found in soil, post-harvest wastes, water, and the air.
Pathogenic soil fungus can live in soil for extended periods of time in the
absence of host plants. They are retained in the soil and on plant leftovers by
mycelium, sclerotia, post spores, egg spores, and thick-walled spores. The
fungus primarily enters the plant through open wounds (such as scratches,
mechanical damage, caused by other objects, ...). They damage the plant's
vascular cells, rendering them incapable of absorbing water and nutrients from
the substrate. As a result, the symptoms of soil fungal diseases, which cause

3

yellow wilt, stunting, and plant mortality, are sometimes very similar
(Doehlemann et al., 2017b).
2.1.1.1. Overview of Fusarium solani
* Research history
Fusarium is an ascomycete fungus genus initially identified by Link in
1809 as Fusisporium. Members of the genus are abundant and may be recovered
as pathogens, endophytes, and saprophytes from plants and soil all over the
world. Members of the genus are well-known for their abilities as plant
pathogens, although research with native plants and soil in undisturbed places
shows that the number of species not connected with recognized illnesses may
considerably exceed those that are. The majority of species of the genus produce
a variety of secondary metabolites that vary greatly in chemical form, may have
a role in plant disease, and may be regulated in commercial and international
trade. Wollenweber and Reinking divided the genus Fusarium into sections in
the 1930s, including Martiella and Ventricosum, which Snyder and Hansen
combined in the 1940s to produce a single species, Fusarium solani one of nine
Fusarium species they recognized based on morphological traits. F. solani is
currently thought to be a species complex composed of numerous, closely
related and visually indistinguishable "confusing" species with distinct genetic
differences. The F. solani species complex (FSSC) is a pathogenic ascomycetes
group pathogen from soil and rhizosphere that causes vascular wilt or root rot in
over 100 plant species. The pathogenic isolates of F. solani are morphologically
indistinguishable from the non-pathogenic strains. F. solani has been identified
using molecular markers EF1- (translation elongation factor 1-alpha), RPB2 66
(RNA polymerase II second largest subunit), and the ITS rDNA region are
among them (Schroers et al., 2016; Homa et al., 2018; Patel et al., 2020).
* Pathogenic mechanism
4

The infection process of hypocotyls for several phylogenetic species
within the FSSC has been described on their respective host plants (Bywater,
1959; Christou, 1962) (Samac & Leong, 1989). After spore attachment, the
easiest route to invade the host plant is via the stomates. The role of direct
penetration of plant tissue in disease development is uncertain. Passive barriers,
such as cutin, a waxy polymer component of the cuticle, must be overcome to
infect the plant. Variability in the role of cutinase in pathogenesis has been
reported for plant-pathogenic fungi. A number of regulatory elements in the
promoter of cutinases control expression, in particular a palindromic sequence
which binds cutinase transcription factor 1α (CTF1α), which positively regulates
expression in the presence of cutin (Kämper et al., 1994); (Li & Kolattukudy,
1997); (Li et al., 2002). A second transcription factor, termed CTF1β, is
involved in the constitutive expression of some cutinase genes. The expression
of a FSSC 11 cutinase gene in Mycosphaerella spp., a fungus unable to infect
unwounded papaya fruit, conferred to the transgenic fungus the ability to infect
intact tissue (Dickman et al., 1989). In addition to cutinases, enzymes digesting
pectin in the plant cell wall have been implicated in the penetration of plant
barriers. The enzyme encoded by pelA is induced in the presence of pectin,
whereas the enzyme encoded by pelD is only expressed in planta, where it is
induced by asparagine and homoserine, two amino acids found at high levels in
pea seedlings (Rogers et al., 2000; Yang et al., 2005). Deletion of either pelA or
pelD individually did not cause a significant reduction in virulence; however, a
ΔpelA/ΔpelD mutant was severely impaired in virulence, causing only a few
mild lesions (Rogers et al. , 2000; Coleman, 2016).
* Effects
The fungus Fusarium solani causes the illness, and the initial sign is
yellow leaves that easily break off when lightly shook. The elder leaves drop
first, followed by the higher leaves. The tree was still alive, but the roots were
5

rotting, the root bark had loosened from the wood, and within there were brown
stripes that progressively crept to the taproot. When the illness is severe, all of
the plant's roots rot and it dies. In often flooded locations, the illness frequently
causes catastrophic harm. When the soil is inundated, the roots are deprived of
oxygen, which weakens them. The soil fungus Fusarium solani will readily
attack the root tip, causing the roots to rot. Furthermore, when nematodes sting
in nematode-infested regions, they produce sores, allowing fungus to penetrate
and cause more significant injury. The predominant hosts for Fusarium solani
are potato, pea, bean, and members of the cucurbit family such as melon,
cucumber, and pumpkin.

Some strains may cause infections in humans.

Fusarium damping-off, corn rot, fruit rot, root rot, and surface rot are caused by
Fusarium solani are found in most states in the United States. Fusarium
virguliforme sp. nov., formally known as F. solani f. sp. glycines, causes sudden
death syndrome (SDS) in soybean. The name “sudden death” refers to the early
defoliation and death of the soybean plant. SDS has become a serious problem
in the commercial production of soybeans in North and South America since the
early 1990‟s (Aoki et al., 2003)The first symptoms of root rot in beans are
narrow, long, red to brown lesions on the stems, and lengthwise cracks often
develop. Lesions extend down the main taproot, which may shrivel, decay and
die. The symptoms in some cases extend up the hypocotyl to the soil surface.
Clusters of fibrous roots (lateral roots or adventitious roots) commonly develop
above the shriveled taproot. Severe Fusarium root rot kills primary and
secondary roots of beans, and most times only adventitious roots are visible.
Note the typical redbrown symptoms of Fusarium root rot on the taproot.
Fusarium crown and foot rot of squash and pumpkin is caused by Fusarium
solani f. sp. cucurbitae. The first symptom is wilting of the leaves. Within

several days, the entire plant may wilt and die. If the soil is removed from
around the base of the plant, a very distinct necrotic rot of the crown and upper
6


portion of the taproot can be seen. The rot develops first as a light-colored,
water-soaked area which becomes progressively darker. It begins in the cortex
of the root, causes cortex tissue to slough off, and eventually destroys all of the
tissue except the fibrous vascular strands. Infected plants break off easily about
2-4 cm below the soil line. The fungus generally is limited to the crown area of
the plant. Fusarium dry rot is characterized by an internal light to dark brown or
black rot of the potato tuber-and it is usually dry. The rot may develop at an
injury such as a bruise or cut. The pathogen penetrates the tuber, often rotting
out the center. Extensive rotting causes the tissue to shrink and collapse, usually
leaving a dark sunken area on the outside of the tuber and internal cavities.
Yellow, white, or pink mold may be present (Wharton et al., 2007).
* Prevention
Beans: Planting beans after soils have warmed up (55oF) at a depth of 1/2
inch in coarse, well-drained soil that has been ideally treated is the greatest
preventative approach. A well-prepared seedbed encourages fast seedling
development while reducing root rot. Soil compaction should be reduced, and
hard pans, if present, should be broken up. Bean trash should always be taken to
a location where beans will not be produced for at least 6 years. It is unknown
how long the root rot fungus may persist in the soil; nevertheless, when a 6-year
or longer rotation is used, the disease is kept under control long enough to
generate a lucrative crop.
Squash and Pumpkin: Fusarium crown and foot rot occurs intermittently
in most regions, and the severity of the disease is determined by soil moisture
and inoculum density. Because the fungus only lives in the soil for two to three
years, a four-year rotation is generally enough to keep the illness at bay.

Planting

fungicide-treated

seed

minimizes

disease

contaminated seed.
2.1.1.2. Overview of Colletotrichum gloeosporioides
7

spread

caused

by


* Research history
Colletotrichum gloeosporioides is a ubiquitous pathogen. It belongs to the
order melanconiales. This fungus infects monocotyledons (turf grass) to higher
dicothyledons (cashew trees). C. gloeosporioides is widely distributed and
common plant pathogen in the world (Sutton, 1992); (Cannon et al., 2000). The
fungus is more abundant in tropical and subtropical regions than in temperate
(CAB international 2005). This pathogen infects about 470 different host genera.
The pathogen also causes post-harvest problems (Prusky & Plumbley, 1992) and
also act as endophytic strains which are isolated from symptomless plant parts

(Cannon & Simmons, 2002); (Guozhong et al., 2004); (Photita et al., 2004),
(Photita et al., 2005). C gloeosporioides was proposed for the first time as
Vermicularia gloeosporioides by Penzig in 1882. C. gloeosporioides was first
reported at Deodoro, Brazil in 1937 on S. humilis and in India, it was first
reported by Butler 1918 on coffee. Glomerella cingulata is the sexual stage
(teleomorph) while the asexual stage (anamorph) is called C. gloeosporioides
(Von Schrenk, 1903). There are various species come under genus
Colletotrichum but only C. graminicola and C. higginsianum genomes were
completely sequenced. C. gloeosporioides genome is under study but various
genes have been identified which involve in pathogenesis and host defense
mechanism. It requires 25-280C temperature, pH 5.8-6.5 for better growth. This
pathogen is inactive in dry season and switches to active stages when
encountered favorable environmental conditions. It involves hemibiotrophic
mode of infection where both phases, biotrophic and necrotrophic phases occur
sequentially. Various medium preparations were employed for the growth and
sporulation of C. gloeosporioides including Potato dextrose agar, lima bean
agar, malt extract agar and oat meal agar(Sharma & Kulshrestha, 2015b).
Traditionally the identification and characterization of Colletotrichum spp were
relied on differences in morphology features such as colony color, size, and
8


shape of conidia and appressorium, optimal temperature for growth, growth rate,
presence or absence of setae (Smith & Black, 1990); (Gunnell & Gubler, 1992);
(Sutton, 1992). Now molecular techniques provide alternative methods for
taxonomic studies and are important tools in solving the problem of species
delimitation (Maclean et al., 1993).
* Pathogenic mechanism
Colletotrichum species enter the host plant via wounds, natural openings,
or directly through the appressorium. A large number of Colletotrichum species

penetrate directly, and the formation of the appressorium is required for host
tissue invasion. After infiltrating the host plant, the pathogen may use one of
two colonization strategies: intracellular hemibiotrophy or subcuticular
intramural colonization, or a combination of the two. The intracellular
hemibiotrophy

strategy

is

used

by

Colletotrichum

trifolii

and

C.

lindemuthianum. They form a spherical infection vesicle after penetrating.
Hyphae develop from this vesicle and colonize additional host cells. In this
paradigm, the fungus develops biotrophically at first, without creating
symptoms, but subsequently, during the necrotrophic phase, the fungus causes
host cell destruction, resulting in symptoms (Moraes et al., 2013).
* Effects
C. Gloeosporioides causes anthracnose disease in a variety of crops
around the world. It is a disease that affects the leaves, stems, and fruits of

mango, papaya, guava, custard apple, pomegranate, and other subtropical fruit
crops, causing pre-harvest and post-harvest losses. Anthracnose thrives in damp,
humid, warm weather and is spread by infected seeds, rain splash, and moist
breezes. It commonly leads in fruit drop and fruit rot. Anthracnose is caused by
fungi that produce conidia within black fungal fruiting bodies called acervuli.
Other species are also to blame for the majority of anthracnose illness. First,
lesion appears as small, dark spots on stolons andpetioles. These lesions grow in
9


size as they age. Brownish patches are typically created in a concentric ring
pattern by the conidial masses that cover the lesion site. C. gloeosporioides
infects over 470 different host genera, including avocado, mango, beans,
cassava, citrus plant, cotton, cow-pea, cucumber, eggplant, green gram, mango,
onion, pepper, pumpkin, papaya, sorghum, soybean, tomato, watermelon, wheat,
yam, zucchini/courgette, cucurbit, cereals, legumes, and spinach. Anthracnose
induced by C.gloeosporioides has been documented from all over the world.
Apple bitter rot caused by Glomerella cingulata and C.gloeosporioides has been
documented in North Carolina orchards. The illness was discovered at the end of
June and is expected to cause 100 percent fruit rot by mid-August. C.
gloeosporioides and C. acutatum caused apple and pear fruit rot in the Southern,
Central, and Mid Atlantic areas of the United States, as well as in most other
nations where these fruits are produced (Sharma & Kulshrestha, 2015a).
* Prevention
Non-chemical control: Non-chemical control involves the effective dips of
infected plant or crop in hot water having temperature around 480C for
approximately 20 minutes. This method is not that much effective to eliminate
infection of C.gloeosporioides completely.
Chemical control: Chemical method involves the use of fungicide spray in
orchard having infected plants or crops present. Fungicide spray was not

recommended in rainy season. Fungicide spray applies at the interval of 14-28
days in the orchard is an effective control of this pathogen. There are various
types of fungicides used such as: post-harvest and pre-harvest (Sharma &
Kulshrestha, 2015a).
2.1.2. Over view of plant pathogenic bacteria
Bacteria are able to cause diseases in a wide range of plants throughout
the entire world (Maclean et al. , 1993); (Kannan & Bastas, 2015). These
10


organisms, known as phytopathogenic bacteria, affect all food-producing plants,
colonizing either their surface or tissues (Kannan & Bastas, 2015). They cause
symptoms such as spots, blights, cankers, tissue rots, and/or hormone
imbalances that lead to plant overgrowth, stunting, root branching, and leaf
epinasty, among others (Strange & Scott, 2005); (Kannan & Bastas, 2015).
These issues impact plants on a qualitative and quantitative level, negatively
affecting global food supplies (Kannan & Bastas, 2015). Bacterial diseases of
plants cause devastating damage to crops and significant economic losses.
Collectively, they cause losses of over $1 billion dollars worldwide every year
to the food production chain (Mansfield et al., 2012); (Kannan & Bastas, 2015).
Together with other phytopathogens, such as fungi and viruses, and abiotic
stress factors, including environmental degradation, climate change and
chemical pollution, bacterial phytopathogens pose a global threat to agricultural
food production. Thus, the development and employment of management
approaches to overcome and suppress phytopathogenic bacteria, which includes
mitigating their survival strategies, is imperative to global food security (Strange
& Scott, 2005); (Sundström et al., 2014).
2.1.2.1. Overview of Xanthomonas
* Research history
The first reported observation of a phytopathogenic bacterium that

nowadays would be referred to the genus Xanthomonas was made in 1881 by
Wakker (1883), in the case of the yellows disease of hyacinths. In the century
after Wakker's discovery, a large number of similar bacteria were subsequently
isolated from numerous plant diseases the world over. Given the contemporary
"new" host- "new" species notion and the "Tribe Erwinieae" nomenclatural
concept then in vogue (Lippincott et al., 1981)), phytopathogens of this sort
amounted to dozens of named species of the genus Phytomonas by the date of
the first edition of Elliott's (1930) Manual of Bacterial Plant Pathogens. This
11


genus Phytomonas contained not only these many species of yellow-pigmented,
slime forming, motile, Gram-negative, phytopathogenic, rod-shaped bacteria,
but also numerous species that were nonpigmented or differently pigmented,
many not capable of forming viscous slimes, some that were Gram positive,
others that were nonmotile, etc. The one of these newly named genera that
concerns us here is Xanthomonas Dowson 1939 (Starr, 1981).

* Pathogenic mechanism
Xanthomonas species, one of the most important genera among
phytopathogens, cause diseases in virtually all economically important crops,
including orange, cassava, tomato, pepper, crucifers, cotton, rice, beans, and
grapes. In most of these diseases, the dominant symptoms are necrotic lesions on
foliage, stems, or fruit (spots, streaks, and cankers), wilts (vascular infections),
tissue macerations (rots), or possibly hyperplasias (such as in citrus canker).
Very large numbers of xanthomonad cells, admixed with their characteristic
extracellular slime, are found in the lesions. The copious ooze of this yellow and
sticky mass from the vessels that are exposed by transverse or oblique cuts
across infected stems and petioles constitutes a valuable diagnostic sign in many
vascular infections caused by xanthomonads (Starr, 1981).

* Effects
Xanthomonas axonopodis pv. manihotis (Xam), the causal agent of
cassava bacterial blight, generates losses of up to 100% under appropriate
climatic conditions. This disease threatens food security in the tropics, where
cassava constitutes a major staple food(Medina et al., 2018). Xanthomonas
axonopodis pv. glycines is a phytopathogenic bacterium that causes bacterial
pustule disease in soybean. Globally, this disease causes considerable yield loss
and reduces the quality of the crop. In Korea, the disease affected 89.7% of
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soybean fields in Yeongnam, which resulted in yield losses of 19.8% and 16.8%
in 2006 and 2007, respectively (Moon et al., 2020).
* Prevention
Select cultivars or transplants that are certified free of pathogens.
Choose disease-resistant varieties.
Fumigation of seed beds and soil to kill bacteria.
Clean garden equipment.
Cut down infected plants.
Plow deeply after harvest and bury the remnants of infected plants.
2.1.2.2. Overview of Rastonia solanacearum
* Research history
R. solanacearum is a Gram-negative bacterium with rod-shaped cells, 0.51.5 µm in length, with a single, polar flagellum. The positive staining reaction
for poly-ß-hydroxybutyrate granules with Sudan Black B or Nile Blue
distinguishes R. solanacearum from many other (phytopathogenic) Gramnegative bacterial species. Gram-negative rods with a polar tuft of flagella, nonfluorescent but diffusible brown pigment often produced. On the general
nutrient media, virulent isolates of R. solanacearum develop pearly cream-white,
flat, irregular and fluidal colonies often with characteristic whorls in the centre.
Avirulent forms of R. solanacearum form small, round, non-fluidal, butyrous
colonies which are entirely cream-white. On Kelman‟s tetrazolium and SMSA
media, the whorls are blood red in colour. Avirulent forms of R. solanacearum

form small, round, non-fluidal, butyrous colonies which are entirely deep red
(Osdaghi).
* Pathogenic mechanism
Phytopathogenic bacteria have often developed enzymes to hydrolyze
plant cell wall components to obtain nutrients and energy, which are further
involved in early stages of the infective process, favouring the entry and
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advance of the pathogenic agent in host tissues. R. solanacearum produces
several plant cell wall-degrading enzymes, secreted via the type two secretion
system. These include one β-1,4-cellobiohydrolase (CbhA) and some pectinases
whose activities have been identified respectively as one β-1,4-endoglucanase
(Egl), one endopolygalacturonase (PehA), two exopolygalacturonases (PehB
and PehC), and one pectin methyl esterase (Pme). R. solanacearum Egl is a 43kDa protein that has proved to be involved in pathogenicity. Inactivation of egl,
pehA or pehB genes revealed that each contribute to R. solanacearum virulence,
and a deficient mutant lacking the six enzymes wilted host plants more slowly
than the wild-type. Since pectin catabolism does not significantly contribute to
bacterial fitness inside the plant, it seems that cellulase and pectinolytic
activities are preferably required for host colonization than for bacterial
nutrition. Thus, R. solanacearum hydrolytic enzymes are thought to be involved
in pathogenicity in plantation (Álvarez et al., 2010).
* Effects
The bacterial wilt disease has been described, and the causal agent
isolated, in more than 200 plant species belonging to 53 different botanical
families. The disease has a worldwide distribution (Álvarez et al. , 2010). This
unusually wide host range is continuously expanding, and so descriptions of new
hosts are not uncommon. The most important widespread hosts are banana and
plantain (Musa paradisiaca), eggplant (Solanum melongena), groundnut
(Arachis hypogaea), Heliconia spp., potato (S. tuberosum), tobacco (Nicotiana

tabacum), and tomato (Lycopersicon esculentum). The majority of them mostly
belong to the Solanaceae and Musaceae families. According to host range, R.
solanacearum strains have been classified into five races. Bacterial wilt caused
by Ralstonia solanacearum is one of the major constraints in the production of
economically important crops (Hayward, 1991). It causes severe yield loss
mostly in solanaceous crops as well as in other crops which are grown in
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tropical, subtropical, and temperate regions of the world (Chaudhary et al.,
2017). Yield losses vary up to 91% for tomato, 10%–30% in tobacco, 33%–90%
in potato, 80%–100% in banana, and up to 20% in groundnut (Nion & Toyota,
2015). The pathogen is listed in one among the “Top 10” based on
scientific/economic importance plant pathogenic bacteria in molecular plant
pathology worldwide (Mansfield et al., 2012). One of the reasons for this is that
the R. solanacearum species is composed of a very large group of strains
varying in their geographical origin, host range, and pathogenic behavior. This
heterogeneous group is nowadays recognized as a “species complex” which has
been divided into four main phylotypes (phylogenetic grouping of strains). In
potato alone, it is responsible for an estimated US$1 billion in losses each year
worldwide (Elphinstone, 2005), (Narasimha Murthy et al., 2021).
* Prevention
Chemical methods (pesticides and non-pesticides): Plant disease control
has been largely dependent on the use of pesticides.
Biological control agents: Interest in biological control has increased due
to concerns over the general use of chemicals. The benefits of BCAs are
potentially self-sustaining, spread on their own after initial establishment,
reduced input of nonrenewable resources, and long-term disease suppression in
an environmentally friendly manner.
Organic matter: Organic amendments to soil have direct impacts on plant

health and crop productivity. They are advantageous because they improve the
physical, chemical, and biological properties of soil, which can have positive
effects on plant growth
Physical methods, including biofumigation. A number of physical control
methods, e.g. solarization and hot water treatments, have proved to be effective
against R.solanacearum (Nion & Toyota, 2015).
2.1.2.3. Overview of Clavibacter michiganensis
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