Mollicutes is a class with pliable cell boundary, pleomorphic in shape They are smallest, simplest and self replicating cellular organisms.

1. SPIROPLASMS/MOLLICUTES, TAXONOMY,
SPREAD AND MANAGEMENT
1
Mollicutes class with pliable cell boundary.

2. 2
INTRODUCTION
TAXONOMY
SYMPTOMS
SPREAD
MANAGEMENT
CASE STUDIES
CONCLUSION

3. 3
DOMAIN:
BACTERIA
PHYLUM:
PROTEOBACTERIA
PHYLUM:
FERMICUTES
PHYLUM:
TENERICUTRS
CLASS:
MOLLICUTES
3

4. INTRODUCTION
 Mollicutes is a class with pliable cell boundary,
pleomorphic in shape
 Mycoplasma, phytoplasma and spiroplasma are
prokaryotes lacking cell wall and they belongs to class
Mollicutes (molli-soft and cute-skin).
 They are smallest, simplest and self replicating cellular
organisms.
4
(Thind, 2012)

5. • Discovery and research of mycoplasmas proved difficult due
to their incredibly small size, difficulty in staining and the
challenging laboratory conditions necessary to successfully
culture them.
• Being so minute in size they were not initially identified as
bacteria and were considered viruses for years.
• Later mycoplasmas were confused with the L-forms, which
are bacteria that have lost their cell walls either completely or
partially.
5

6. • 1883 - Louis Pasteur described the first Mycoplasma (now
called Mycoplasma mycoides) as the causal agent of pleuro
pneumonia in Cattle.
• 1889 - Term mycoplasma was first used by A. B. Frank. To
describe an altered state of plant cell cytoplasm by
infiltration of fungus like organisms.
• 1898 - Nocard and Roux isolated and cultured first and
called it as pleuro pneumonia like organisms (PPLO).
• 1929 - Julien Nowak used the term mycoplasm
taxonomically for the bovine Pleuropneumonia organism.
S. A. Hall, 1983
Roux
Nocard
Pasteur
Conrad et al., 1973
MYCOPLASMA
6
A. B. Frank

7. Phytoplasma
 1967 - Phytoplasmas were discovered by Japanese scientists
• Doi and Ishiie in japan reported agents resembling mycoplasma
in the sieve tubes while studying with mulberry dwarf disease
• They termed them as mycoplasma-like organisms or MLOs.
 From 1967 to 1994 the phytopatogenic mollicutes are referred as
MLOs
 1994 – the working team on MLOs of the international
Organisation of Mycoplasmology proposed to the subcommittee
on taxonomy of mollicutes that the term phytoplasma replace
the trivial term MLO.
7

8. SPIROPLASMA
Saglio et al. (1971) and Fudl-Allah et al. (1971) succeeded in
culturing the MLO’s, causing citrus stubborn.
The organisms were wall less, helical and motile filaments.
In 1972, Davis et al., observed similar helical, wall less, motile
filaments from the phloem sap of corn stunt diseased maize
plants.
Saglio et al., (1973) described the new genus Spiroplasma.
8

9. CHARACTERISTICS OF
MOLLICUTES
9

10. MOLLICUTES
Lack cell wall, Pleomorphic.
Incapable of synthesis of peptidoglycan.
Surrounded by unit membrane.
Among mollicutes phytoplasmas are not culturable .
Mycoplasma and spiroplasma are culturable and they produce
colonies appear like fried egg.
Resistant to penicillin and its derivatives and sensitive to lysis
by osmotic shock, detergents, alcohols, and specific antibody
plus complement.
10

11. “Fried egg" colonial morphology.
11
MYCOPLASMA

12. Lack flagella
Genome sizes range from 580 to 2200 kbp, among the
smallest recorded in prokaryotes.
The G+C content of the DNA is usually low, ∼23–34 %.
Three organelles: cell membrane, ribosomes, and a
circular double stranded DNA .
Phytoplasma and spiroplasm reproduce in the
haemolymph of insects before travelling to salivary glands.
12

13. MYCOPLASMA
The name Mycoplasma, from the Greek mykes (fungus) and
plasma (formed).
They are filterable, usually non motile.
Reproduction - binary fission.
These require sterols in the form of cholesterol for the stability of their
cytoplasmic membrane and other complex media consisting of beef
extract infusion, peptone, yeast extract and serum with various
supplement.
The different strains vary in their growth rate and may take from two
days to several weeks to form a colony.
13

14. PHYTOPLASM
 Transmits via Grafting, dodder, and insects.
 Insects – Leafhopper, Plant hopper, Psyllids (survive and
replicate).
 These are non cultivable pathogens.
 Intracellular; Obligate parasites.
14

15. Based on molecular data, new taxonomy and designation has
evolved as “Candidatus” phytoplasma species.
Genome size ranges from 530 kb to 1185 kb.
The codon UGA works as a stop codon.
Known to cause more than 600 diseases in several hundred
plant species.
They cause important losses for the national economies,
reaching 70-100% in most of crops of economic interest.
15

16. Spiroplasma
Characters:
Helical branched filamentous mollicutes & Size: 100-
240 nm in dia.
Can be cultured on nutrient media, appear like fried
egg appearance.
Helical filamemts are motile, move by a slow
undulation of filaments.
Colonies on agar medium are 0.2 mm in dia.
Transmitted by insects .
16

17. TAXONOMY
17

18. PHYLOGENY
• The first comparative phylogenetic analysis of the origin of
mollicutes was carried out by oligonucleotide mapping of
16S rRNA gene sequences (Woese et al., 1980).
18

19. • Based on the phylogeny of 16S rRNA genes, the class Mollicutes was
included in the phylum Firmicutes in the most recent revision of
the Taxonomic Outline of Bacteria and Archaea.
• However, the Mollicutes are excluded from the most recently
emended description of the Firmicutes based on alternative
phylogenetic markers, including
19
1. RNA polymerase subunit B,
2. Chaperonin GroEL,
3. Aminoacyl tRNA synthetases, and
4. Subunits of F0F1-ATPase
(Garrity et al., 2007)
(De Vos et al., 2009)

20.  An analysis of 16S rRNA gene sequences is now mandatory
for characterization of novel species.
 The modern species concept for mollicutes is justified by
DNA–DNA hybridization and serology.
20
(Brown et al., 2007)
Characterization of new species.

21. Taxonomy
Domain Bacteria
Phylum Tenericutes
Class Mollicutes
21

22. TAXONOMY AND CLASSIFICATION OF
MOLLICUTES
MOLLICUTES
ACHOLEPLASMATALES
ANAEROPLASMATALES
ENTOMOPLASMATALES
MYCOPLASMATALES
ACHOLEPLASMATACEAE
ANAEROPLASMATACEAE
ENTOMOPLASMATCEAE
SPIROPLASMATACEAE
MYCOPLASMATACEAE
ACHOLEPLASMA
PHYTOPLASMA
ANAEROPLASMA
ASTEROLEPLASMA
ENTOMOPLASMA
MESOPLASMA
MYCOPLASMA
UREAPLASMA
SPIROPLASMA
Bergeys manual of systematics of archaea and
bacteria, 2015 22
INCERTAE SEDIS

23. 23
Order Family Genus Species Genome size
range (kbp)
Habitate Defining
features
MYCOPLASMATACEAE Mycoplasmsa 116,9,1,4 580-1350 H,A -
MYCOPLASMATACEAE Ureaplasma 7,0,0,0 760-1140 H,A Urea
hydrolysis
INCERTAE SEDIS Eperythroozoon 4,0,0,0 Nd A Hemotropic
INCERTAE SEDIS Haemobaronella 1,0,0,0 Nd A Hemotropic
ENTOMOPLASMATCEAE Entomoplasma 6,0,0,0 870-900 N,P -
ENTOMOPLASMATCEA Mesoplasma 11,0,0,0 825-930 N,P Growth with
PES
SPIROPLASMATACEAE Spiroplasma 37,0,0,0 780-2220 N,P Helical
morphology
ACHOLEPLASMATACEAE Acholeplasma 18,0,0,0 1500-1650 A,N,P -
INCERTAE SEDIS Candidatus
Phytoplasma
0,27,0,0 530-1350 N,P Not yet
cultured
ANAEROPLASMATACEAE Anaeroplasma 4,0,0,0 1500-1600 A Strictly
anaerobe
ANAEROPLASMATACEAE Asteroplasma 1,0,0,0 1500 A Strictly
anaerobe
(Edward and Freundt, 1967)
Valid Candidatus Incertae sedis Invalid

24. The Candidatus concept
For a valid description of a bacterial species, it has to be
cultivated.
Some bacteria (Phytoplasma’s) in nature cannot be
cultivated but such bacteria can be characterised by
molecular methods ex. 16S rRNA gene sequencing.
Murray and Schleifer (1994) proposed the rank or
category of Candidatus for classification of such
prokaryotes.
24
(Razin and Hermann, 2002)

25. International Committee on Systematic Bacteriology
accepted the proposal in 1995.
There after Candidatus concept has been introduced to
describe prokaryotic taxa having molecular data but not
the characters required for description according to
Bacteriological code.
Ex. ‘Candidatus Phytoplasma’
25
(Razin and Hermann, 2002)

26.  Domain : Bacteria
 Phylum : Tenericutes
 Class : Mollicutes
 Order : Acholeplasmatales
 Family : Incertae sedis
 Genus : ‘Candidatus Phytoplasma’
Position of phytoplasma inTaxonomy

27. Rules for the description of organisms as novel
taxa within ‘Ca. Phytoplasma’
1. The ‘Ca. Phytoplasma’ species description should refer to a single, unique 16S
rRNA gene sequence (>1200 bp).
2. A strain can be described as a novel ‘Ca. Phytoplasma’ species if
its 16S rRNA gene sequence has <97.5% similarity to that of any
previously described ‘Ca. Phytoplasma’ species.
3. The rank of subspecies should not be used
4. The reference strain should be made available to the scientific
community from the authors of the Candidatus species
description paper
5. Manuscripts that describe a novel ‘Ca. Phytoplasma’ species
should preferably be submitted to the International Journal of
Systematic and Evolutionary Microbiology (IJSEM)
The IRPCM Phytoplasma/Spiroplasma Working Team, 2004

28. 6. The abbreviation for Candidatus is Ca.
7. Phytoplasmas that share >97.5% of their 16S rRNA gene
sequence, but clearly represent ecologically separated populations
and therefore, may deserve description as separate species
• For such cases, description of two different species is recommended only
when all three of the following conditions apply:
(i) The two phytoplasmas are transmitted by different vectors
(ii) The two phytoplasmas have a different natural plant host
(iii) There is evidence of significant molecular diversity, achieved
by either hybridization to cloned DNA probes, serological
reaction or PCR-based assay
The IRPCM Phytoplasma/Spiroplasma Working Team, 2004

29. Candidatus phytoplasma description
Character Description Reference
Morphology Single unit membrane, pleomorphic Doi et al. (1967)
Habitat Phloem sieve, gut, haemolymph of
sap sucking insects
Tsai et al. (1979)
Antibiotic
sensitivity
Tetracycline Ishii et al. (1967)
DNA base
composition
G+C : 23-29% Kollar & Seemuller
(1989)
Genome 530-1350 bp Neimark & Kirkpatrick
(1993)
Codon usage UGA- stop codon, not for tryptophan Lims & Sears (1991)
Sterol in
membrane
Non sterol requiring Lim et al. (1992)
Ribosomal RNA Two rRNA operons & a spacer 16s -
23s rRNA genes
Kuske & Kirkpatrick
(1992)
Phytoplasma/Spiroplasma Working Team IRPCM (2000) IJSEM 29

30. DIAGNOSIS
Before molecular techniques were developed, the diagnosis of
phytoplasma diseases was difficult because they could not be
cultured.
Visual symptoms.
Treating infected plants with antibiotics-tetracycline.
Molecular diagnostic techniques for the detection of
phytoplasma- ELISA based methods.
In the early 1990s, PCR-based methods were developed that
were far more sensitive than those that used ELISA and RFLP
analysis allowed the accurate identification of different strains
and species of phytoplasma.
30

31.  Ultrathin sections of the phloem tissue from suspected
phytoplasma infected plants would also be examined for
their presence.
 Diene’s stain- it is more specific. Phytoplasma stained by
this stained blue. Healthy phloem will be clear of colour
(Deeley et al., 1979).
 DAPI (4,6-diamidino-2 phenylindole); a nucleic acid
specific stain is used. It stains the bacteria in the phloem
sieve tubes (Sinclair, 1989).
31

32. Identification of non-culturable pathogens
Methods
Serological tests, e.g. ELISA
Electron microscopy
Molecular tests, e.g. PCR
32

33. 33
Scanning Electron Micrograph of Arecanut Palm
Phytoplasma.
Healthy tissues of sieve tube of
root
Diseased tissues
Rajeev et al. (2010)

34. Transmission Electron Micrograph of Arecanut Palm
Phytoplasma
Rajeev et al. (2010)
Phytoplasma
Phytoplasma
34

35. Genome sequence is already completed for
1) ‘Candidatus Phytoplasma Onion Yellows M’ (OY-M)
(Oshima et al., 2004).
2) Asters yellow witches broom(AY-WB) (Bai et al., 2006).
3) ‘Candidatus Phytoplasma asteris’ a strain of ‘Candidatus
Phytoplasma australiense’ (Nguyen et al., 2008).
4) ‘Ca. P. mali’ (Kube et al., 2008). Etc…
35
Genome sequencing
(Phyllis et al., 2010)

36. Bermuda grass phytoplasma has the
smallest genome of 530 kb.
The genomes of aster yellows phytoplasmas,
which are the largest (1185 kbp) among the
phytoplasmas.
36
(Phyllis et al., 2010)

37. 37
(Phyllis et al., 2010)

38. (Lee et al., 2000)
Phyllody
Witches broom
Yellowing
Little leaf
Proliferation
Virescence
Stunting
Purple top
Phloem necrosis
38

39. Sugarcane Grassy Shoot Coconut lethal yellowing
39

40. Periwinkle Phyllody Rose Phyllody
Tomato phyllody Trifoliate Phyllody Rice Yellow Dwarf
40

41. Lethal yellowing
Sesame phyllody Little leaf of brinjal
Carrot yellow
Witches’ broom lime Palm wilt Brassica phyllody
41
Aster Yellows

42. Phytoplasma associated with Ajwain
(Trachyspermum ammi)
Samad et al.(2010)
Yellowing, little leaf, curling and stunted growth
42

43. Sandal spike
43
Sandal spike was the first phytoplasma disease reported in India
(Varma et al., 1969).

44. Sesame phyllody
Floral proliferation
Phyllody symptoms
Cracked capsules
Floral virescence
Yellow, Twisted, Reduced leaves
44

45. Important diseases in India
Disease Host Area First Report of etiology
Little leaf Brinjal
Periwinkle
All India
Lukhnow
Varma et al. (1969)
Rao et al. (1983)
X disease Peach NE region Ahlawat & Chenulu (1979)
Bushy Stunt Brinjal New Delhi Mitra & Chakraborty (1988)
Phyllody Bottle gourd &
other gourd
Black pepper
Sesame
Banglore
Banglore
Kerala
All India
Sastry & Singh (1981)
Bhat et al. (2006)
Sahambi (1970)
Witches’ broom Acid Lime
Winged bean
Sunhemp
MH,AP Ghosh et al. (1999)
Singh (1991)
Sharma et al. (1990)
Rubbery wood Citrus Darjeeling Ahlawat & Chenula (1985)
Root wilt Coconut Kerala Solomon et al. (1983)
Sandal spike Sandal Kerala,Kr Varma et al. (1969)
GSD Sugarcane All India Rishi et al. (1973)
White leaf Bermuda grass UP Rishi (1978) Rao et al. (2007)
Yellow dwarf Rice All India Raychaudhri et al. (1967)
45

46. phytoplasma disease Report
Phyllody of Parthenium hystrophorus Pathak (1975)
White leaf disease of Cynodon dactylon Singh et al. (1978)
Bottle guard Phyllody Singh and Singh (1978)
Yellowing disease of Urtochloa panicoides Muniyappa et al. (1982)
Tomato marginal flavenscence Singh (1987)
Phonix sylvestris Pundir (1987)
Soyabean phyllody Sangeta (1991)
Spear rot complex of oil palm Kochu Babu (1993)
Chili little leaf Singh and Singh (2000)
Witch broom in Citrus Ghosh and Singh ( 2002)
Little leaf disease of Pigeon pea
(Candidatus Phytoplasma asteris)
Raj ( 2005)
Medicago sativa,N.tabaccum, Verbesina encliode Ahir ( 2006)
Sida acuata Gaur and Verma (2006)
Yellow crown of Mesta Sardar (2006) 46

47. Little leaf disease of Portulaca grandiflora Ajay Kumar et al.(2007)
Cassia tora and Cleome icosandra Rangaswamy (2007)
Witch broom on Acacia auricduliformi Pardeep Kumar (2007)
Phytoplasma on Mung bean. Narendra Singh(2008)
Little Leaf Disease of Psyllium (Plantago ovata) Samad (2008)
'Candidatus Phytoplasma trifolii‘associated with Little
leaf of Datura inoxia Chrysanthemum morifolium,
Adenium obesum, and Gladiolus
Raj( 2008)
Witches’ broom and Little leaf disease of Zinnia elegans Agrahari ( 2009)
Phyllody of Anise (Pimpenella anisum) Sharma (2009)
Little leaf disease in Rosa alba, Catharanthus roseus,
and Hibiscus rosasinensis
Chaturvedi et al. (2009)
Candidatus phytoplasma asteris’ infecting banana Raj et al.( 2009)
Phyllody and witch broom in Acaranthus hispidium,
Tephrosia purpurea, Passiflora edulis.
Bhale (2010)
Leaf yellows of Calotropis gigantea Priya (2010)
'Candidatus Phytoplasma asteris‘ associated with green
ear disease of bajra
Kumar (2010)
47

48. DISTRIBUTION
Phylogenetic group Distribution Reference
Aster yellows (16SrI) America, Europe, Asia, Africa Lee et al. (2004)
Peanut witches’-broom (16SrII) America, Africa, Europe, Asia,
Australia
Zreik et al. (1995)
X-disease(16SrIII) America, Europe, Asia Davis et al. (2013)
Coconut lethal yellowing (16SrIV) America, Africa Harrison et al.(2002)
Elm yellows (16SrV) Europe, America, Asia, Africa Jung et al. (2003)
Clover proliferation (16SrVI) Europe, America, Asia Hiruki & Wang (2004)
Ash yellows(16SrVII) America, Europe Griffith et al. (1999)
Loofah witches’-broom (16SrVIII) Asia Ho et al. (2001)
Pigeon pea witches’-broom
(16SrIX)
Europe, Asia, America Verdin et al. (2003)
Apple proliferation (16SrX) Europe, America Seemuller & Schneider
(2004)
48

49. Rice yellow dwarf (16SrXI) Europe, Asia, Africa Jung et al. (2003)
Stolbur (16SrXII) Europe, Asia, America,
Africa, Australia
Davis et al. (1997)
Mexican periwinkle virescence
(16SrXIII)
America Gundersen et al. (1997)
BGWL (16SrXIV) Europe Marcone et al. (2004)
Hibiscus witches’ broom (16SrXV) America Montano et al. (2001)
Sugarcane yellow leaf syndrome
(16SrXVI)
America Arocha et al. (2005)
Papaya bunchy top (16SrXVII) America Lee et al. (2006)
American potato purple top wilt
(16SrXVIII)
America Lee et al. (2006)
Chestnut witches’ broom
(16SrXIX)
Asia Jung et al. (2002)
Rhamnus witches’ broom
(16SrXX)
Europe Marcone et al. (2006)
Pinus phytoplasmas (16SrXXI) Europe Schneider et al. (2005)
Lethal yellow disease
Mozambique(16SrXXII)
- Harrison et al.(2014)
49

50. Buckland valley grapevine
yellows(16SrXXIII)
- Wei et al. (2007)
Sorghum bunchy shoot(16SrXXIV) - Wei et al. (2007)
Weeping tea witches
broom(16SrXXV)
- Wei et al. (2007)
Sugarcane phytoplasma
D3T1(16SrXXVI)
- Wei et al. (2007)
Sugarcane phytoplasma
D3T2(16SrXXVII)
- Wei et al. (2007)
Derbid phytoplasma(16SrXXVIII) - Wei et al. (2007)
Cassia witches’ broom (16SXXIX) Asia Al-Saady et al. (2008)
Salt cedar witches’ broom (16SXXX) Asia Zhao et al. (2009)
Soybean stunt (16SXXXI) America Lee et al. (2011)
Malaysian periwinkle virescence and
phyllody (16SXXXII)
Asia Nejat et al. (2012)
Allocasuarina muelleriana
phytoplasma (16SXXXIII)
Australia Marcone et al. (2003)
Bertaccini et al. (2014)50

51.  Domain : Bacteria
 Phylum : Tenericutes
 Class : Mollicutes
 Order : Entomoplasmatales
 Family : Spiroplasmataceae
 Genus : Spiroplasma
Position of Spiroplasma in taxonomy

52. Citrus stubborn:
• Disease was first noticed by E. R. Waite in 1915 on navel
orange trees in california and named as stubborn
• Fawcett et. al., 1994 first showed the transmissible nature.
• A MLOs in the sievetubes of stubborn infected citrus tissue
was discovered independently by Igwegbe and calavan (1970)
in california and by Lafleche and Bove (1970) in France.
• Both groups of workers concluded that a mycoplasma, and not
a virus, was probably cause.
52

53. • Saglio et. al., (1971) in France and Fudl-Allah
et. al., (1972) in California were able to culture
a MLO’s in liquid and solid media.
• The organism was described and named
Spiriplasma citri (saglio et. al., 1973).
53

54. • From 1915-1917 several non-productive trees were
observed and some were top-worked using carefully
selected healthy buds.
• In spite of the carefully chosen budwood, the growth of the
scion buds was slow and exhibited growth characteristics
shown by the original tree so its called stubborn.
• This disease also called little leaf was thought to have been
the result of drought. Subsequent work showed many
similarity between little leaf and stubborn and present
both names apply to the same disorder.
54
Karl Maramorosch and
Raychaudhri, 1988

55. SYMPTOMS:
More or less gradual yellowing.
 Smaller leaves.
 Shortened internodes.
 Excessive proliferation of leaves.
 More or less rapid dieback,
decline, and death.
 Root abnormalities and necrosis.
 Low quality fruits un marketable.
55
The infected tree is stunted and has
compact growth.

56.  Rind of the fruits will be dense
 Premature dropping.
 Unpleasant flavored and taste.
o Pathogen: Spiroplasma citri
Geographic Distribution:
 USA, Mediterranean,
Middle East, North Africa
Insect Vectors:
 Circulifer tenellus and
 Neoaliturus haematoceps
Circulifer tenellus
several infected fruit showing
delayed coloration and
reduced size, infected leaves
showing mottling,
56

57. CORN STUNT DISEASE
 Corn stunt occurs in the southern United States, Central
America, and northern South America.
 Early symptoms consist of yellowish streaks in the
youngest leaves.
 Later, much of the leaf area turns reddish purple.
 Infected plants remain stunted.
 Infected plants often have more ears, but the ears are
smaller and bear little or no seed.
57

58.  The corn stunt pathogen is Spiroplasma kunkelli .
 It is transmitted in nature by the leafhoppers Dalbulus
elimatus and D. maidis.
 The leafhoppers must feed on diseased plants for several
days before they can acquire the spiroplasma,
 Incubation period of 2 to 3 weeks
 Plants show corn stunt symptoms 4 to 6 weeks after
inoculation.
58

59. Extreme stunting and yellowing
Spiral cells of S. kunkelii in a phloem cell of an infected corn plant.
59

60. Corn stunt spiroplasma isolated from infected corn plants and grown media.
(A)Typical helical morphology of spiroplasma.
(B) Active spiroplasmas from liquid culture observed by dark-field microscopy.
(C) Colonies of corn stunt spiroplasma on agar plates 14 days after inoculation
60

61. SPREAD
1. Graft transmission
2. Dodder transmission
3. Seed transmission
4. Insect transmission
61

62. Graft Transmission and Dodder Transmission
 Coleman (1917): Sandal spike
Pal & Pushkarnath (1935): Sesame phyllody
Varma ( 1969): Little leaf Brinjal
Jha (1973): GSD
62

63. Seed transmission
• Coconut lethal yellowing
• Alfalfa witches broom
• Seeds from phytoplasma-infected lime (Citrus aurantiaca) and
tomato (Lycopersicum esculentum) from Oman and Italy
respectively were allowed to germinate under sterile
conditions, and tested at several growth stages
(Bertaccini and Duduk, 2009)
63

64. Insect transmission
 Sahambi (1970): transmission of Sesame Phyllody by Orosius albicinictus.
 Sinha (1984): leaf hopper transmission (Paraphlepsius irroratus) of Aster
yellow
 Srivastava et al.(2006): transmission of GSD by leafhopper
Deltocephalus vulgaris
• Phytoplasmas are mainly spread by insects in the families
Cicadellidae (leafhoppers)
Fulgoridae (planthoppers)
Psyllidae (psyllids)
Leafhopper, Plant hopper and Psyllid
64

65. Insect transmission
 Phytoplasmas may overwinter in insect vectors or perennial
plants.
 Phytoplasmas enter the insect's body through the stylet, move
through the intestine, and are then absorbed into the
haemolymph.
 From here they proceed to colonise the salivary glands, a process
that can take up to three weeks.
 Once established, phytoplasmas will be found in most major
organs of an infected insect host.
65

66. 66

67. MANAGEMENT
67

68. Management
Roguing
Field sanitation
• Removal of inoculum sources, infected crop debries, etc.
Pathogen-free propagation materials
Vector Control
• Methyldemeton 25 EC 2 ml/litre
• Dimethoate 30 EC 2 ml/litre
• Malathion 50 EC 2 ml/litre
68

69. Using antibiotics
• Tetracyclines are bacteriostatic to phytoplasmas, they
inhibit their growth.
• However, without continuous use of the antibiotic, disease
symptoms will reappear. Thus, tetracycline is not a viable
control agent in agriculture.
Resistance variety:
• Rice yellow dwarf -CH-2, CH-45, CH-126-33-11, IR-127-80-
1-10, MR-278.
• Little leaf of brinjal - Nurki, Hisar Shyamal and H-10. Pusa
Purple Long, Pusa Purple Round, Pusa Purple Cluster
69
Muniyappa and Ramakrishnan (1976)
Sidhu and Dhatt (2007)

70. SESAMUM PHYLLODY
 Increase in nitrogen application, reduce the phyllody.
 Resistant varieties RJS78, RJS147, KMR14, KMR79, Pragati,
IC43063 and IC43236 ,Rajeswari (highly adoptable female
parent).
 Resistant sources
• Sesamum alatum and
• Sesamum mulayanum
 Spray 2-3 times with monocrotophos(0.03%) or Dimethoate (0.2
%) at flowering stage.
 Spray 500ppm tetracyclin @ flowering.
70
Singh et al. (2007)

71. Sugacane grassy shoot (GSD)
• Using of disease free setts.
• Remove and burn the infected clumps periodically.
• Avoid ratooning in problem area.
• moist heat treatment at 540c for 30 min. Shukla and Singh
(1990)
• Control vector by spraying malathion and Dimethoate @ 2
ml/lit.
71

72. INTEGRATED MANAGEMENT OF ROOT WILT OF COCONUT
 Follow integrated nutrient management
 Apply organic manure @ 50kg / palm / year
 Apply balanced dose of chemical fertilizers i.e. 500g N, 300g
P, 1000g K in two splits 1/3rd during April-May and 2/3rd
during Sep-Oct. under rainfed condition
 Four splits – Jan., April, July and Oct.- irrigated
 1kg Magnesium sulphate also has to be applied along with
second dose of fertilizer application

73. Cut and remove disease advanced, uneconomical palms yielding <
10 nuts / palm /year
Provide adequate drainage facilities
Grow green manure crops cowpea, sunhemp near coconut basins
during April-May and incorporated during September-October
Irrigate with at least 250 litre / week
Adopt suitable inter/mixed cropping
Coconut development board

74. Citrus stubborn
i. Antibiotics
ii. Clean propagative material
iii. Quarantine
iv. Vector control
v. Roguing: Removing stubborn affected trees
from young orchards.
vi. Top working should be done only on trees
which are totally free of stubborn.
74

75. What is new in the Field?
Phytoplasma colonies under optical microscope [(a) magnification 50×,
(b) magnification 20×]

76. Methodology was patented under the application number PCT/IB2012/052965 on June
12, 2012.
Phytoplasma successful cultured on Axenic media
PivS and PivL (Phytoplasma in vitro solid/liquid medium)
Periwinkle shoots infected with phytoplasmas.
Shoot were moistened with 0.5 mL of PivL with phenol red, liquid plus plant pieces
were transferred to a 4 mL vacuette tube (incubation at 25±1°C)
Colour change from orange-red (pH above 7.0) to yellow (pH below 6.8) which could
indicate growth of phytoplasmas
50 μL of acid broth cultures were inoculated onto 6 cm diameter plastic plates
containing 8 mL of PivS
incubated in an atmosphere of 5% CO2 and 95% N2 for 5‒7 days at 25±1°C
Phytoplasma colonies under binocular microscope
PCR assays using phytoplasma specific primers confirmed that phytoplasma DNA
Identification of phytoplasma using RFLP analysis and direct sequencing
of selected amplicons

77. Acid colour change (lefthand tube) Righthand tube
inoculated at the same time with healthy
micropropagated periwinkle at 15 days after
inoculation
Phytoplasma colonies under optical microscope: 12x, 20 and 50× magnification
Contaldo et al., 2012

78. CASE STUDY
78

79. 100 leaf hoppers 100 plants
01 02 03 04 05 06 07 08 09 10
01
02
03
04
05
06
07
08
09
10
F
I
E
L
D
G
L
A
S
S
H
O
U
S
E

80. Plant 1 Plant 2
Experiment 1 Experiment 2
Spiroplasma and phytoplasma
were detected by PCR in both
plant 1 and plant 2
Transmission experiment
Isolate X * Isolate Y *

81. Experiment 1 (inoculum from plant 1)
1 2 3 4 5 6 7 8 9 10
11 12 13 14 15 16 17 18 19 20
1 2 3 4 5 6 7 8 9 10
S + P S + P S + P S + P S + P S + P S + P S + P S + P S + P
20 replications
OBSERVATION FROM RANDOMLY SELECTED 10 PLANTS 60 DAS
•All plants showed at least one reddish leaf and chloratic stripes
•All plants showed positive for PCR analysis

82. Experiment 2 (inoculum from plant 2)
1 2 3 4 5 6 7 8 9 10
11 12 13 14 15 16 17 18 19 20
1 2 3 4 5 6 7 8 9 10
P P S + P
20 replications
OBSERVATION FROM RANDOMLY SELECTED 10 PLANTS 60 DAS
only 3 plants showed reddish leaves

83. CONCLUSION
From Exp. 1 :
1. The consistent transmission of both phytoplasmas
and spiroplasmas from plant 1 suggest surviving
adaptation (together) of these pathogens (P + S).
From Exp. 1 & 2 :
1. Two isolates exhibited different symptoms.
2. Two Isolates of mollicutes have differential
transmission rate at same environmental conditions.
Oliveira et al., 2015

84. Integrated management of sesame phyllody
during kharif
An integrated disease management trail was taken up
to evaluate the effect of various treatments on incidence of
sesame phyllody. Sowing of imidacloprid treated sesame
cultivar E-8, was taken up on July 2010 and two foliar
sprays of different treatments were taken up at 30 DAS
and 60 DAS.
84
CASE STUDY
Reddy, 2011

85. T1 - ST with Imidachloprid @ 5g/kg followed by Clothionidin spray @0.5 g/l
T2 - ST with Imidachloprid @ 5g/kg followed by Imidachloprid spray @0.3 ml/l
T3 - ST with Imidachloprid @ 5g/kg followed by Verticillium lecanii spray @0.3 ml/l
T4 - ST with Imidachloprid @ 10g/kg followed by Acephate spray @1g/l
T5 - ST with Imidachloprid @ 5g/kg followed by without spray
T6 - ST with Imidachloprid @ 5g/kg followed by Nimbicidin spray @0.5%
T7 - ST with Imidachloprid @ 5g/kg followed by Tetracycline spray @ 250 ppm
T8 – Untreated check
Treatments
Reddy, 2011

86. Per cent disease incidence
Sl . No. Per cent incidence after 1st
spray
Per cent incidence after 2nd
spray
T1 16.66 46.66
T2 18.33 36.66
T3 21.66 51.66
T4 21.66 43.33
T5 28.33 58.33
T6 31.66 51.66
T7 33.33 63.33
T8 43.33 73.33
S.Em 2.36 1.46
CD (5%) 7.17 4.44
Reddy, 2011
Sl . No. Per cent incidence after 1st
spray
Per cent incidence after 2nd
spray
T1 16.66 46.66
T2 18.33 36.66
T3 21.66 51.66
T4 21.66 43.33
T5 28.33 58.33
T6 31.66 51.66
T7 33.33 63.33
T8 43.33 73.33
S.Em 2.36 1.46
CD (5%) 7.17 4.44
ST with Imidachloprid @ 5g/kg followed by
Imidachloprid spray @0.3 ml/l
T8 – Untreated check

87. CONCLUSION
• The result revealed that
• T2 has recorded lowest percent disease
incidence in both sprays (18.66% and 36.66%)
followed by T4 (21.66% & 43.33%) as
compared to untreated check (43.33% and
73.33%) and other treatments.
87
T2 - ST with Imidachloprid @ 5g/kg followed by Imidachloprid spray @0.3 ml/l
T4 - ST with Imidachloprid @ 10g/kg followed by Acephate spray @1g/l
Reddy, 2011

88. Research conducted in
UASD
88

89. Studies on phytoplasma disease of
periwinkle
89
Sanjeev kumar, 2010

90. Incidence of phytoplasma disease of
periwinkle during 2009
Sanjeev kumar,2010

91. Micrograph of ultrthin section of leaf midrib
showing phytoplasmal bodies in periwinkle
Sanjeev kumar,2010

92. 92
Sridhar daman, 2012
Studies on phytoplasma with special
reference to sesamum phyllody

93. Survey of sesamum phyllody in Northern
Karnataka
Sridhar Daman,2012
Sesamum phyllody average incidence in various districts
of Northern Karnataka during kharif 2009

94. Insect transmission of sesamum phyllody Dodder transmission of sesamum phyllody
Transmission of sesamum phyllody
Sridhar Daman,2012

95. STUDIES ON LITTLE LEAF OF BRINJAL CAUSED
BY ‘Candidatus Phytoplasma trifolii’
95
Rathnamma (2014)

96. Little leaf disease incidence on
different cultivars of brinjal
Rathnamma,2014

97. Confirmation of little leaf of brinjal by amplification of
16S rRNA gene
Rathnamma,2014

98. Conclusion…
98

99. Thank You...
99