Regarding diseases caused by Agrobacterium, Erwinia and xanthomas

1. University of Agricultural Sciences,GKVK, Bengaluru
Department of Plant Pathology
PAT 512 (2+1)
Diseases of vegetable and spice crops
PRESENTED BY:
MADHU.J
PAMB 1321
Jr.Msc plant pathology

2. HEAT
FLODDING
UV LIGHT
FREEZING
DROUGHT
SALINITY NEMATODES
ANIMALS
BACTERIA
VIRUSES
FUNGUS
INSECTS
WEEDS
ABIOTIC STRESS BIOTIC STRESS

3. Xanthomonas
• Xanthomonas is a genus of bacteria, many of which cause plant
diseases.
• There are at least 27 plant associated Xanthomonas spp., that all
together infect at least 400 plant species.
• Different species typically have specific host and/or tissue range and
colonization strategies.

4. • Xanthomonas species can cause bacterial spots and blights of leaves,
stems, and fruits on a wide variety of plant species. Pathogenic species
show high degrees of specificity and some are split into multiple pathovars,
a species designation based on host specificity.
• Citrus canker, caused by Xanthomonas citri subsp. citri is an economically
important disease of many citrus species (lime, orange, lemon, pamelo).

5. • Bacterial leaf spot has caused significant crop losses.This disease
include Xanthomonas vesicatoria and Xanthomonas perforans =
[Xanthomonas axonopodis (syn. campestris)
pv. vesicatoria], Xanthomonas vesicatoria.

6. • Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae, is a
disease found worldwide and particularly destructive in the rice-
producing regions in Asia.
• Xanthomonas campestris pv. punicae cause bacterial blight of
pomogranate.

8. Morphology and growth
Colony growth characteristics include:
• Mucoid, convex, and yellow colonies on YDC medium
• Yellow pigment from xanthomonadin, which contains bromine
• Most produce large amounts of extracellular polysaccharide
• Temperature range – 4 to 37 °C, optimal growth 25-30 °C

9. Taxonomy
KINGDOM
PHYLUM
CLASS
ORDER
FAMILY
GENUS
Bacteria
Pseudomonadota
Gammaproteobacteria
Xanthomonadales
Xanthomonadaceae
Xanthomonas
SPECIES campestris
Individual cell characteristics include:
Cell type – straight rods
Size – 0.4 – 1.0 µm wide by 1.2 – 3.0 µm long
Motility – motile by a single polar flagellum

10. X. citri X. citri pv. citri Citrus Citrus canker
X.
citri pv. fuscans
Bean (Phaseolus vulgaris L.) Bacterial blight X. citri pv. fuscans
X.
citri pv. glycines
Soybean (Glycine max) Bacterial pustule X. citri pv. glycines
Xanthomonas
spp. Pathovar Host Disease
X. oryzae X. oryzae pv. oryzae Rice Bacterial blight
X. oryzae pv. oryzicola Rice Bacterial streak
X. phaseoli X. phaseoli pv. phaseoli Bean (Phaseolus
vulgaris L)
Bacterial blight
X. translucens X. translucens pv. translucens Wheat Black chaff
X. translucens pv. undulosa Wheat Black chaff
X. vasicola X. vasicola pv. vasculorum Sugarcane Gumming disease
Xanthomonas Species

11. X. citri pv. punicae Pomegranate (Punica
granatum)
Leaf blight X. citri pv. punicae
X. euvesicatoria X.
campetris pv. vesicato
ria
Pepper and tomato Bacterial leaf spot
X.
citri pv. malvacearum
Cotton (Gossypium
spec.)
Bacterial blight X.
citri pv. malvacearum
Xanthomonas spp. Pathovar Host Disease
X. campestris pv.
raphani
Brassica oleracea Bacterial leaf spot X. campestris pv.
raphani
X. campestris pv. vitia
ns
Lettuce Bacterial leaf spot X. campestris pv. vitia
ns
X. arboricola X. arboricola pv.
punicae
Pomegranate (Punica
granatum)
Leaf blight
X. axonopodis X. axonopodis pv. ma
nihotis
Cassava Bacterial blight
X. campestris X.
campestris pv. armor
aciae
Horseradish Bacterial leaf spot
X. campestris pv. ca
mpestris
Brassicaceae Black rot

12. BACTERIAL SPOT ON TOMATO
Xanthomonas campestris pv. vesicatoria
• Due to the wide diversity within the bacterial spot disease, it can cause
disease in different temperatures and has become a threat to tomato
production worldwide.

13. Symptoms
• Spots are generally dark brown to black and circular (but can be angular)
on the leaves.
• Disease usually starts on the lower leaves, and symptoms are initially
more visible on the underside of the leaves.
• Leaf lesions are initially circular and water-soaked, later turning dark
brown to black with a wet or greasy appearance.
• A developing lesion may have a faint halo, which eventually disappears.

14. • When conditions are optimal for disease development, primary lesions
can coalesce to form irregularly shaped lesions and areas of extensive
necrosis.
• A general yellowing and blighting may occur on leaflets with many
lesions.
• Bacterial spot lesions can also develop along leaf margins as the
pathogen infects leaf hydathodes, resulting in blackening of the leaf
margins and foliar blighting.
• Dead foliage will often remain on the plant, which can give it a scorched
appearance.
• Fruit lesions begin as small, slightly raised blisters. As spots increase in
size, they become dark brown, scab-like, and slightly raised.

16. Black rot: Xanthomonas campestris pv. campestris
• The infection of the foliage results in yellow ‘V’ shaped spots arising
along the margin which extend in the direction of the midrib.
• These spots are associated with a typical black discoloration of the
veins.
• The infection extends through the xylem to the stalk and the vascular
bundles turn black.
• In severe infection, the whole leaf shows discoloration and eventually
falls off.

17. Mode of Spread and Survival
• Black rot is spread rapidly during warm, humid weather, with an optimal
temperature range of 27- 30°C at 80- 100% humidity. Once in the soil, the
bacteria are spread by splashing rain and wind. Bacteria enter plants
through wounds or natural openings at the leaf margins called hydathodes

18. Management
• Use of certified disease-free seeds and transplants
• Hot water treatment of non-certified seeds; chemical treatments with
sodium hypochlorite, hydrogen peroxide, and hot cupuric acetate or zinc
sulfate may also be used
• Crop rotation with non-cruciferous plants (3–4 years)
• Removal of crop debris after harvest
• Control of cruciferous weeds that may serve as reservoir for the pathogen
• Sanitation (e.g., clean equipment, avoiding work in wet fields, etc.)
• The development and use of black rot resistant cultivars the most
common and potentially useful sources of black rot resistance occur in
other brassica genomes including B. rapa, B. nigra, B. napus, B.
carinata and B. juncea.
• Seed treatment with Aureomycin 1000ppm for 30 min is effective in
killing both the internally and externally seed-borne pathogen. Drenching
the nursery soil with formaldehyde 0.5% helps in checking the disease.
Application of bleaching powder at 10.0 to 12.5 kg/ha controls the
disease.

19. Role of effectors in plant disease
• Pathogenicity of Xanthomonas and most other Gram-negative
phytopathogenic bacteria depends on a conserved type III secretion
(T3S) system which injects more than 25 different effector proteins into
the plant cell.
• Extensive studies on the molecular mechanisms of type III effector
function revealed that effector proteins with enzymatic functions seem to
play important roles in the interaction of Xanthomonas with its host
plants, for example, the SUMO protease XopD.
• In addition, xanthomonads express a unique class of type III effectors to
pursue another strategy.
• Effectors of the AvrBs3 family, so far only identified in Xanthomonas
spp. and Ralstonia solanacearum, mimic plant transcriptional activators
and manipulate the plant transcriptome.

20. • Pathogenicity of Xanthomonas campestris pathovar (pv.) vesicatoria
and most other Gram-negative bacterial plant pathogens largely depends
on a type III secretion (TTS) system which is encoded by hypersensitive
response and pathogenicity (hrp) genes.
• These genes are induced in the plant and are essential for the bacterium
to be virulent in susceptible hosts and for the induction of the
hypersensitive response (HR) in resistant host and non-host plants.
• The TTS machinery secretes proteins into the extracellular milieu and
effector proteins into the plant cell cytosol. In the plant, the effectors
presumably interfere with cellular processes to the benefit of the
pathogen or have an avirulence activity that betrays the bacterium to the
plant surveillance system.
• A number of effector proteins are members of families, e.g., the AvrBs3
family in Xanthomonas.
• AvrBs3 localizes to the nucleus of the plant cell where it modulates plant
gene expression.
• Another family that is also present in Xanthomonas is the YopJ/AvrRxv
family.
• The latter proteins appear to act as SUMO cysteine proteases in the host.

21. Erwinia…..
• Erwinia is a genus of Enterobacterales bacteria containing mostly plant
pathogenic species which was named for the famous plant pathologist,
Erwin Frink Smith.
• It contains Gram-negative bacteria related to Escherichia coli, Shigella,
Salmonella, and Yersinia.
They are primarily rod-shaped bacteria.
✦Rod shaped, Motile
✦Gram negative Bacteria
✦Non sporing, Facultative anaerobes

22. Taxonomy
• Erwinia amylovora
• Erwinia aphidicola
• Erwinia billingiae
• Erwinia chrysantum
• Erwinia mallotivora
• Erwinia papayae
• Erwinia persicina
• Erwinia psidii
• Erwinia pyrifoliae
• Erwinia rhapontici
• Erwinia toletana
• Erwinia tracheiphila
• Erwinia carotovora
Species
KINGDOM
PHYLUM
CLASS
ORDER
FAMILY
GENUS
Bacteria
Proteobacteria
Gammaproteobacteria
Enterobacteriales
Enterobacteriaceae
Erwinia
SPECIES carotovora

23. E. amylovora, which causes fire blight on apples, pears, and other Rosaceae crops
E. tracheiphila, though, causes bacterial wilt of cucurbits.
E. Carotovora causing soft rot of vegetables – black leg of potato,

24. BACTERIAL SOFT ROT
• Bacterial soft rots occur worldwide and cause serious diseases of crops in
the field, in transit, and especially in storage.
• They cause a greater total loss of produce than any other bacterial
disease.
• Bacterial soft rots reduce quantities of produce available for sale, reduce
the quality and thus the market value of crops, and increase expenses
greatly for preventive measures against soft rots.

25. • Bacterial soft rots occur most commonly on fleshy storage tissues
of vegetables and annual ornamentals such as potatoes, carrots,
onions, iris, and fleshy fruit such as cucumber and tomato, or
succulent stems, stalks, or leaves, such as cabbage, lettuce, celery,
and spinach.
• In the tropics, soft rots often develop on the fleshy stems of some
plants while still in the field, e.g., in corn, cassava, and banana.
• Nearly all fresh vegetables are subject to bacterial soft rots, which
may develop within a few hours in storage or during marketing.

26. Symptoms
• Soft-rot symptoms begin as a small water-soaked lesion, which enlarges
rapidly in diameter and in depth.
• The affected area becomes soft and mushy while its surface becomes
discolored and somewhat depressed.
• Tissues within the affected region become cream colored and slimy,
disintegrating into mass of disorganized plant cells and bacteria.
• The outer surface may remain intact while the entire contents have
changed to a turbid liquid; alternatively, cracks develop and the slimy
mass exudes to the surface and, in air, turns tan, gray, or dark brown.

27. • A whole fruit or tuber may be converted into a sort, watery, decayed
mass within 3 to 5 days.
• Infected fruits and tubers of many plants are almost odorless until they
collapse, and then secondary bacteria grow on the decomposing tissues
and produce a foul odor.
• When root crops are affected in the field, the lower parts of the stem
may also become infected and watery and may turn black and shrivel,
causing the plants to become stunted, wilt, and die.

28. • Erwinia caratovora pv. atroseptica, the cause of blackleg of potato, may
be thought of as a cool temperature variant of E. caratovora pv.
carotovora and is restricted mostly to potatoes.
• The Pathogens Erwinia chrysanthemi affects many hosts and causes
many of the soft rot of tropical plants while they are still growing in the
field.
• Soft-rot bacteria can grow and are active over a range of temperatures
from 5 to 35°C.
• They are killed with extended exposure at about 50°C.

29. Development of Disease
• Soft-rot bacteria survive in infected fleshy organs in storage and in the
field, in debris, on roots or other parts of host plants, in ponds and
streams used for water irrigation, occasionally in the soil, and in the
pupae of several insects.
• The disease may first appear in the field on plants grown from
previously infected seed pieces.
• Some tubers, rhizomes, and bulbs become infected through wounds or
lenticels after they are set or formed in the soil.
every 20 to 60 minutes
18° and 35°C

30. • The inoculation of bacteria into fleshy organs and their further
dissemination in storage and in the field are facilitated greatly by
insects.
• Soft-rot bacteria can live in all stages of the insect. Moreover, the bodies
of the insect larvae(maggots) become contaminated with bacteria when
they crawl about on rotting seed pieces, carry them to healthy plants,
and put them into wounds where they can cause the disease.
• Even when the plants or storage organs are resistant to soft rot and can
stop its advance by the formation of wound cork layers, the maggots
destroy the wound cork as fast as it is formed and the soft rot continues
to spread. When soft-rot bacteria enter wounds, they feed and multiply
at first on the liquids released by the broken cells on the wound surface.
• There they produce increasing amounts of pectolytic enzymes that
break down the pectic substances of the middle lamella and bring about
maceration of the tissues.

31. Disease Cycle

32. MANAGEMENT
• Use of certified disease-free seeds.
• Examine seed tubers for any soft rot symptoms before planting.
• Avoid excessive irrigation and prevent standing water in fields. if possible,
irrigate with well water and not surface water Balanced fertilization and
increase the calcium content in soil.
• Rotate with non-solanaceous crops in the following year and eliminate
volunteer potatoes that arise from previous season leftovers
• Storage in ventilated stores at low temperatures (5°C) and clean storage
areas with formaldehyde or copper sulphate.

33. Control
• The control of bacterial soft rots of vegetables is based almost
exclusively on sanitary and cultural practices.
• All debris should be removed from warehouses, and the walls should be
disinfested with formaldehyde or copper sulfate.
• Wounding of plants and their storage organs should be avoided as much
as possible.
• Products to be stored should be dry, and the humidity and temperature
of warehouses should be kept low.

34. Control In the field,
• plants should be planted in well-drained areas and at sufficient
distances to allow adequate ventilation.
• Susceptible plants should be rotated with cereals or other
nonsusceptible crops.
• Few varieties have any resistance to soft rot, and no variety is immune.
• Chemical sprays are generally not recommended for the control of soft
rots.
• Control of the insects that spread the disease reduces infections both in
the field and in storage.
• Biological control of bacterial soft rot of potatoes has been obtained by
treating potato seed pieces before planting with antagonistic bacteria or
with plant growth-promoting rhizobacteria.

35. Proteases
Cellulases
Pectinases
Hydrolyses of
gelatin and pectin
Virulence
factors
Nucleases
Lipases
Starch is not hydrolyzed
Optimum temperature for Erwinia carotovora ranges from 27-30°C
maximum varies from 32-40°C.
Bacterium generally produces white colonies on nutrient agar and cloudy
growth in nutrient broth.

36. Agrobacterium

37. Agrobacterium
• Agrobacterium is a genus of Gram-negative bacteria established by H. J.
Conn that uses horizontal gene transfer to cause tumors in plants.
• Agrobacterium tumefaciens is the most commonly studied species in
this genus.
• It is famous for taking advantage of its host by injecting DNA derived
from its Ti plasmid into the host causing the plant to create galls which
excrete opines .
• Agrobacterium tumefaciens is the causal agent of crown gall in over 140
species of eudicots.
• Agrobacterium tumefaciens was first isolated from grapevine galls in
1897.
• Symptoms are caused by the insertion of a small segment of DNA from a
plasmid, into the plant cell, which is incorporated at a random location
into the plant genome.
• Economically, A. tumefaciens is a serious pathogen of walnuts, grape
vines, stone fruits, nut trees, sugar beets, horse radish.

38. • Soil bacterium which induces crown gall disease
• Gram negative rods
• Enters easily through plant wounds Also forms tumours.
• Although no naturally occuring plasmids are known in higher plants, one
bacterial plasmid, the Ti plasmid of A. tumefaciens is of great
importance.
white colony appearance ,convex
colonies with a pearly lustre

39. Nature’s
smallest genetic
engineer
Taxonomy
KINGDOM
PHYLUM
CLASS
ORDER
FAMILY
GENUS
Bacteria
Pseudomonadata
Alfaproteobacteria
Hypomicrobiales
Rhizobiaceae
Agrobacterium
SPECIES tumifaciens
These bacteria are Gram-negative
and grow aerobically, without
forming endospores.
The cells are rod-shaped and
motile, having one to six
peritrichous flagella

40. • Transformation – the process of obtaining transgenic plants Transgenic plant
– a plant with a foreign gene (or genes) from another plant/animal that is
incorporated into its chromosome.
• the gene transfer mechanism between Agrobacterium and plants,which
resulted in the development of methods to alter the bacterium into an
efficient delivery system for genetic engineering in plants.
• Most common genes (and traits) in transgenic or biotech crops herbicide
resistance Insecticide resistance Bt genes in field corn (maize) virus-
resistance (coat-protein) genes.

41. Infection And Pathogenesis
1. Signal induction to Agro bacterium:
2. Attachment of Agro bacterium to plant cells
3. Production of virulence proteins.
4. T – DNA Transfer And Integration
5. Production of T-DNA strand
6. Transfer of T-DNA out of Agro bacterium
7. Transfer of T-DNA into plant cells and integration

42. Methods of Gene Transfer
Direct or vectorless gene transfer
• Chemical mediated gene transfer: Certain chemicals like polyethylene
glycol (PEG) and dextran sulphate induce DNA uptake into plant
protoplasts.
• Microinjection
• Electroporation Methods of Gene Transfer
• Liposome mediated method of Gene Transfer
• Biolistics
Indirect or vector – mediated gene transfer

43. Indirect or Vector-Mediated Gene Transfer
• Gene transfer is mediated with the help of a plasmid vector is known as
indirect or vector mediated gene transfer.
• Among the various vectors used for plant transformation, the Ti-plasmid
from Agrobacterium tumefaciens has been used extensively.
• This bacterium has a large size plasmid, known as Ti plasmid (Tumor
inducing) and a portion of it referred as T-DNA (transfer DNA) is
transferred to plant genome in the infected cells and cause plant tumors
(crown gall).
• Since this bacterium has the natural ability to transfer T-DNA region of
its plasmid into plant genome, upon infection of cells at the wound site,
it is also known as the natural genetic engineer of plants.

44. Natural genetic engineer of plants
• The ability to cause crown gall disease is associated with the presence
of the Ti (tumor inducing) plasmid within the bacterial cell.
• This is a large (greater than 200 kb) plasmid that carries numerous
genes involved in the infective process
• A remarkable feature of the Ti plasmid is that, after infection, part of the
molecule is integrated into the plant chromosomal DNA
• This segment, called the T-DNA, is between 15 and 30 kb in size,
depending on the strain.
• It is maintained in a stable form in the plant cell and is passed on to
daughter cells as an integral part of the chromosomes

45. • plasmid is that the T-DNA contains eight or so genes that are expressed
in the plant cell and are responsible for the cancerous properties of the
transformed cells.
• These genes also direct synthesis of unusual compounds, called opines,
that the bacteria use as nutrients.
• In short, A. tumefaciens genetically engineers the plant cell for its own
purposes.

47. Symptoms and signs
• Crown gall, disease of plants caused by
Agrobacterium tumefaciens
• 1000’s of plant species are susceptible; they
include especially rose, nut trees, grape, many
shrubs and vines and perennial garden plants
• Symptoms:
• The galls, at first cream coloured or greenish,
later turn brown or black.
• Galls are roundish, rough-surfaced galls, several
inches or more in diameter, usually at or near the
soil line, or on roots and lower stems white or
flesh coloured (young stage)
• Galls become hard and corky on woody stems,
knobby and knotty
• Affected plants stunted with chlorotic leaves.

48. Disease Cycle and Epidemiology
• Agrobacterium tumefaciens naturally resides on the woody and
herbaceous weeds.
• Its presence in soils originates from galls that were broken or sloughed
off from infected plants during cultivation practices or disseminated as
infected plant material.
• Irrigation aids in further dissemination of the A. tumefaciens bacterial
cells.
• Agrobacterium tumefaciens is also spread by infected and infested
planting stocks originating as nursery stock from uncertified sources.

49. • Secondary spread then occurs through pruning and cultivation
equipment, particularly when galls are removed manually with the same
cutting tools used in pruning.
• Tilling equipment can be contaminated by cutting through galls at or
near the base of trunks of infected trees.
• Rogueing (removal) of infected trees and replanting in the same spot
where the infected tree had grown is poor practice because sloughed
off galls serve as sources of abundant populations of A. tumefaciens.

50. Management
• Preplanting Management Options
Selection of good Planting stocks.
Proper Site selection for planting.
Crop rotation with cereal crops followed by green manuring.
Chemical eradicants-copper-based solutions,sodium hypochlorite.
Biological control-Dipping the root system into a suspension of A.
radiobacter K84 before planting in infested fields.
Genetically engineering. Transgenic crop plants harboring one or more
unique genes tailored to protect the plant from crown gall have been
developed.
• Management in Established Fields
• The removal of infected trees and vines is costly in loss in time and in
money.
• Usually, rogueing of the diseased plants is sufficient to minimize further
spread of crown gall.

51. • Regulatory measures
• Crop rotation with maize or other grain crops
• Avoid injuries to roots or lower stem parts
• Penicillin or vancomycin - partial control
• Agrobacterium radiobacter (Strain K1026) (No gall) applied to
fresh wounds.
• controlling root-chewing insects and nematodes
• cutting away large galls on trees, and disinfecting the wounds.