Molecular detection and identification of phytoplasmas in sugarcane in Hawaii, Thailand, Cuba and Near East

Molecula de ec ion and iden i ica ion o phy oplasmas in
suga cane in Hawaii, Thailand, Cuba and Nea Eas
Disse a ion
Zu E langung des G ades Dok o de Na u wissenscha en
-D . e . na .-
de Fakul ä ü Biologie, Chemie und Geowissenscha en
de Uni e si ä Bay eu h
o geleg on
Ziad Sou i
Aus La akia, Sy ien
Bay eu h, Ge many
Janua 2012
Die o liegende A bei wu de in de Zei on Juli 2007 bis Janua 2012 am Leh s uhl ü
P lanzenphysiologie de Uni e si ä Bay eu h un e de Lei ung on He n P o . D . Ewald
Komo ange e ig .
Volls ändige Abd uck de on de Fakul ä ü Biologie, Chemie und Geowissenscha en
de Uni e si ä Bay eu h genehmig en Disse a ion zu E langung des akademischen
G ades eines Dok o s de Na u wissenscha en (D . e . na .).
Disse a ion einge eich am: 11. 01. 2012
Wissenscha liches Kolloquium: 27. 03. 2012
Am ie ende Dekanin: P o . D . Bea e Lohne
P ü ungsausschuss:
P o . D . Ewald Komo (E s gu ach e )
P o . D . Ha old L. D ake (Zwei gu ach e )
P o . D . Ge ha d Rambold (Vo si z)
P o . D . Klaus H. Ho mann
PD D . S e an Geime
I
Con en s
1. In oduc ion ....................................................................................................... 1
1.1. Plan diseases ............................................................................................................................... 1
1.2. Phy oplasmas as plan pa hogens ................................................................................................ 2
1.3. De ini ion o phy oplasmas .......................................................................................................... 2
1.4. T ansmission and sp ead o phy oplasmal diseases .................................................................... 6
1.4.1. Hos cycle o phy oplasmas ................................................................................................... 6
1.4.2. Hos Speci ici y o Phy oplasmas .......................................................................................... 8
1.5. Phylogene ic posi ion o phy oplasmas ....................................................................................... 8
1.6. Managemen and con ol o phy oplasma diseases .................................................................... 9
1.6.1. P e en ion s a egy ................................................................................................................ 9
1.6.2. Con ol o insec ec o s ...................................................................................................... 10
1.6.3. Managemen s a egy .......................................................................................................... 10
1.7. Ana omy o phloem cells ............................................................................................................ 10
1.8. Some phy oplasma diseases o suga cane ................................................................................. 11
1.8.1. Suga cane yellow lea synd ome ......................................................................................... 11
1.8.2. Suga cane whi e lea and suga cane g assy shoo ............................................................... 12
1.9. De ec ion o suga cane phy oplasma in ec ions ........................................................................ 13
1.10. Suga cane yellow lea synd ome in Hawaii .............................................................................. 14
2. Ma e ial and Me hods ...................................................................................... 17
2.1. Ma e ial ...................................................................................................................................... 17
2.1.2. Chemicals and Enzymes ...................................................................................................... 17
2.1.2.1. Chemicals ..................................................................................................................... 17
2.1.2.2. Enzymes ....................................................................................................................... 18
2.1.3. Bu e s, Solu ions ................................................................................................................ 18
2.1.3.1. Bu e and solu ions o DNA ex ac ion ..................................................................... 18
2.1.3.2. Bu e o gel elec opho esis ....................................................................................... 19
2.1.3.3. Bu e o PCR ............................................................................................................. 19
2.1.3.4. Bu e o es ic ion enzymes ...................................................................................... 19
2.1.3.5. Bu e o polyac ylamide gel elec opho esis ............................................................. 19
2.1.4. Ki s ...................................................................................................................................... 20
2.1.4.1. Isola ion o Nucleic Acids o PCR .............................................................................. 20
2.1.4.2. Nucleic acids pu i ica ion ............................................................................................. 20
II
2.1.4.3. Q-PCR .......................................................................................................................... 20
2.1.5. Oligonucleo ides .................................................................................................................. 21
2.1.6. So wa e o Gene analysis .................................................................................................. 21
2.2. Me hods ..................................................................................................................................... 22
2.2.1. Plan ma e ial ....................................................................................................................... 22
2.2.2. DNA ex ac ion s a egies ................................................................................................... 22
2.2.3. Polyme ase Chain Reac ion (PCR) o he de ec ion o phy oplasmas ............................... 24
2.2.3.1. De ini ion o Nes ed PCR ............................................................................................. 24
2.2.3.2. Nes ed PCR Reac ion ................................................................................................... 25
2.2.3.3. Fi s ound o PCR
.................................................................................................... 26
2.2.3.4. Nes ed ound o PCR .................................................................................................... 26
2.2.3.5. Nes ed-PCR assay (I) ................................................................................................... 26
2.2.3.6. Nes ed-PCR assay (II) .................................................................................................. 27
2.2.3.7. Nes ed-PCR assay (III) ................................................................................................. 27
2.2.3.8. Nes ed-PCR assay (IV) ................................................................................................ 28
2.2.4. Aga ose Gel Elec opho esis ............................................................................................... 29
2.2.5. Diges ion o nes ed-PCR p oduc s ...................................................................................... 30
2.2.5.1. Inac i a ion o es ic ion enzymes .............................................................................. 30
2.2.6. Polyac ylamide Gel Elec opho esis ................................................................................... 31
2.2.6.1. S eps o ope a ion ......................................................................................................... 31
2.2.6.2. Special equipmen ........................................................................................................ 31
2.2.6.3. De ec ion o DNA in polyac ylamide gels by s aining ................................................. 31
2.2.7. Sequencing and phylogene ic analysis o ibosomal DNA ................................................. 31
2.2.7.1. Sample P epa a ion o Value Read Se ice in Tubes ................................................. 32
2.2.8. Ho wa e ea men ............................................................................................................. 32
2.2.8.1. P epa a ion o he plan ma e ial p io o he ho wa e ea men ................................ 33
2.2.9. Suga cane aphid ansmission es ....................................................................................... 33
2.2.9.1. Insec ea ing ................................................................................................................ 33
2.2.9.2. Plan ma e ial ................................................................................................................ 33
2.2.9.3. T ansmission es s ........................................................................................................ 34
2.2.10. Q-PCR ( eal- ime PCR) assay ........................................................................................... 34
2.2.10.1. Me hods o moni o ing DNA ampli ica ion in qPCR ................................................ 34
2.2.10.2. De ec ion o phy oplasma based on TaqMan qPCR assays........................................ 36
2.2.11. T ansmission Elec on Mic oscopy (TEM) ....................................................................... 37
III
2.2.11.1. P epa a ion o hin sec ions ........................................................................................ 38
3. Resul s ............................................................................................................. 39
3.1. Es ablishmen o he es o phy oplasma ................................................................................ 39
3.1.1. PCR o de ec ion o phy oplasma ...................................................................................... 39
3.1.2. Sou ces o suga cane samples ............................................................................................. 41
3.2. Phy oplasma in suga cane in Hawaii, Cuba, Egyp and Sy ia ..................................................... 42
3.2.1. Phy oplasma de ec ion by nes ed-PCR assay (I) and iden i ica ion by RFLP ..................... 42
3.2.2. Phy oplasma de ec ion by nes ed-PCR assay (II) and iden i ica ion by RFLP ................... 46
3.2.3. Phy oplasma de ec ion by nes ed-PCR assay (III) .............................................................. 48
3.2.4. Phy oplasma de ec ion by nes ed-PCR assay (IV) and iden i ica ion by RFLP .................. 49
3.3. Phy oplasma in suga cane in Hawaiian plan a ions (2009) ....................................................... 52
3.3.1. Phy oplasma de ec ion and iden i ica ion ............................................................................ 54
3.4. Phy oplasma in suga cane in Hawaiian o me plan a ion ields (2009) ................................... 55
3.4.1. Phy oplasma de ec ion and iden i ica ion ............................................................................ 56
3.5. Phy oplasma in suga cane in Hawaiian b eeding s a ion (2010) ............................................... 57
3.5.1. Phy oplasma de ec ion and iden i ica ion ............................................................................ 57
3.6. Phy oplasma in suga cane in Hawaiian b eeding s a ion and plan a ions (2011) ..................... 58
3.6.1. Phy oplasma in suga cane in Hawaiian plan a ion .............................................................. 58
3.6.1.1. Phy oplasma de ec ion and iden i ica ion ..................................................................... 59
3.6.2. Phy oplasma in suga cane in Hawaiian b eeding s a ion (2011) ........................................ 61
3.6.2.1. Phy oplasma de ec ion by nes ed-PCR assay (II) and iden i ica ion ............................ 61
3.6.2.2. Phy oplasma de ec ion by nes ed-PCR assay (III) and (IV) and iden i ica ion ............ 63
3.6.3. Phy oplasma in suga cane in di e en a eas close o o me plan a ions ............................ 67
3.6.3.1. Phy oplasma de ec ion and iden i ica ion ..................................................................... 67
3.6.4. Phy oplasma in g ass weeds in Sumida wa e c ess a m ................................................... 69
3.7. Phy oplasma in suga cane in Thailand (2010 – 2011) ................................................................ 70
3.7.1. Phy oplasma in suga cane in Bang Ph a and Khon Kean p o inces (2010) ....................... 70
3.7.1.1. Phy oplasma de ec ion and iden i ica ion ..................................................................... 71
3.7.2. Phy oplasma in suga cane in Suphan Bu i p o ince (2011) ................................................ 75
3.7.2.1. Phy oplasma de ec ion and iden i ica ion ..................................................................... 75
3.8. Es ablishmen o TaqMan qPCR assay as ano he es o phy oplasma ................................... 76
3.8.1. Pe o mance cha ac e is ics o qPCR .................................................................................. 76
3.8.1.1. E iciency Measu emen ............................................................................................... 77
3.8.1.2. A i icial samples o es sensi i i y o qPCR assay ..................................................... 81

IV
3.8.2. Q-PCR esul s o he suga cane samples om di e en sou ces ........................................ 83
3.8.3. Dis ibu ion o phy oplasma in suga cane ........................................................................... 88
3.10. Ho wa e ea men in o de o ge phy oplasma ee suga cane plan ................................ 96
3.10.1. Ho wa e ea men acco ding o Aus alian ecipe .......................................................... 96
3.10.2. Ho wa e ea men wi h a ious du a ion ........................................................................ 97
3.11. Insec icide ea men o phy oplasma-in ec ed suga cane plan ............................................ 99
3.12. T ansmission es wi h suga cane aphid (Melanaphis saccha i) ............................................ 100
3.13. T ansmission elec on mic oscopy o cy ological loca ion o phy oplasma ......................... 101
3.13.1. Ana omy o lea phloem issue ........................................................................................ 101
3.13.2. Localiza ion o phy oplasma in ec ion ............................................................................ 102
3.13.3. Phy oplasma size and shape ............................................................................................ 106
3.13.4. Ul as uc u al changes o he phy oplasma in ec ion on lea e ana omy ........................ 108
4. Discussion ..................................................................................................... 110
4.1. Es ablishmen o he es o phy oplasma .............................................................................. 110
4.1.1. E iciency o PCR ampli ica ion ....................................................................................... 110
4.1.2. Ca y-o e con amina ion p oblems .................................................................................. 112
4.1.3. Q-PCR ( eal- ime PCR) ..................................................................................................... 112
4.2. Phy oplasma de ec ion and iden i ica ion by RFLP analysis .................................................... 115
4.2.1. Phy oplasma ypes in Hawaiian and Cuban suga cane ..................................................... 115
4.2.2. Phy oplasma ypes in Thai suga cane ................................................................................ 117
4.2.3. Phy oplasma ypes in Egyp ian and Sy ian suga cane ...................................................... 118
4.3. Iden i ica ion o he phy oplasma s ains in suga cane by phylogen ic analysis ..................... 120
4.4. Ho wa e ea men in o de o ge phy oplasma ee plan ................................................. 121
4.5. T ansmission es wi h suga cane aphid .................................................................................. 122
4.6. T ansmission elec on mic oscopy o cy ological loca ion o phy oplasma ........................... 123
5. Summa y ....................................................................................................... 125
6. Zusammen assung ......................................................................................... 127
7. Acknowledgemen ......................................................................................... 129
8. Re e ences ..................................................................................................... 130
9. Lis o igu es ................................................................................................ 145
10. Lis o ables ................................................................................................ 148
E klä ung ........................................................................................................... 150
V
Abb e ia ions
AAY Ame ican as e yellows
AY as e yellows
bp base pai
BSA bo ine se um albumin
°C deg ee Celsius
C h eshold cycle
CTAB hexadecyl ime hylammonium b omide
dd double dis illed
dNTP deoxy ibonucleo ide iphospha e
DNA deoxy ibonucleic acid
EDTA E hylenediamine e aace ic acid
FAM 6-ca boxy luo escein
Fig igu e
GS g assy shoo
HWT Ho wa e ea men
kb kilo base pai s
l li e
µ mic o
n-PCR nes ed- polyme ase chain eac ion
OY onion yellows
PCR polyme ase chain eac ion
PVP poly inylpy olidone
Q-PCR Quan i a i e PCR
qPCR Quan i a i e PCR
RNA ibosomal RNA
RFLP es ic ion agmen leng h polymo phism
RNase ibonuclease
RNA ibosomal RNA
VI
pm e olu ions pe minu e
RT oom empe a u e
RYD ice yellow dwa
S second
SCYLP suga cane yellow lea phy oplasma
SCYLV suga cane yellow lea i us
SCWL suga cane whi e lea
SCGS suga cane g assy shoo s
SGS so ghum g assy shoo s
TAMRA 6- e ame hyl hodamine
TBE T is base- Bo ic acid- EDTA
TEM T ansmission Elec on Mic oscopy
RNA T ans e RNA
U uni
UV Ul a iole
YLS yellow lea synd om
In oduc ion
1
1. In oduc ion
1.1. Plan diseases
“Plan s make up he majo i y o he ea h’s li ing en i onmen as ees, g ass, lowe s, e c.
Di ec ly o indi ec ly, plan s also make up all he ood on which humans and all animals
depend. Plan s a e he only highe o ganisms ha can con e he ene gy o sunligh in o
s o ed, usable chemical ene gy in ca bohyd a es, p o eins, and a s” (Ag ios, 2004).
“Plan diseases a e e y impo an pa o plan p o ec ion wi hin he sys em o plan /c op
p oduc ion. Diseases can signi ican ly lessen he g ow h and yield o educe he u ili y o a
plan o plan p oduc . Heal hy plan s g ow and unc ion o he maximum o hei gene ic
po en ial. Howe e , plan s a e conside ed o be diseased when hey a e nega i ely a ec ed by
a disease-causing agen ha lead o in e e ing wi h hei no mal de elopmen and
physiological unc ions” (Ag ios, 2004).
“Co ec diagnosis o he cause o a disease is an essen ial s ep in o de o cons uc a
con enien s a egy o manage he plan disease. Usually, he i s s ep includes he
de e mina ion o he p obable cause o he disease: whe he he disease is caused by an
in ec ious agen (pa hogen) o en i onmen al ac o . Since diseases in plan s a e caused by
ei he non-li ing (abio ic, non-pa asi ic, non-in ec ious, ‘non-pa hogenic’) en i onmen al
ac o s o li ing (bio ic, pa asi ic, pa hogenic, in ec ious) agen s. On he o he hand, plan
diseases a e g ouped based on he causal agen in ol ed (de iciency diseases, ungal diseases,
bac e ial diseases, i al diseases, mollicu es diseases, e c.), he plan pa a ec ed o he ype
o symp oms” (Ag ios, 2004).
In gene al, plan disease is any g ow h o de elopmen al condi ion ha is no “no mal” o ha
plan and can usually diminish i s economic o aes he ic alue. In many cases, he plan
pa hologis s depend on symp oms and signs o he disease o hypo he ical diagnosis o
diseases in plan s. The cha ac e is ic in e nal o ex e nal al e a ions showed by he plan in
eac ion o he disease-causing agen a e called symp oms.
Plan pa hology is he s udy o he pa hogens and o he en i onmen al ac o s ha cause
disease in plan s, and he me hods o p e en ing o con olling disease and educing he
damage i causes. Uncon olled plan diseases may esul in less ood and highe ood p ices,
o in low-quali y ood (Ag ios, 2004). O e he las decades scien is s in molecula plan
pa hology ha e also es ablished a new se o diagnos ic ools and echniques ha a e used o
In oduc ion
8
1.4.2. Hos Speci ici y o Phy oplasmas
Plan hos ange o a phy oplasma is dependen upon ec o s speci ici y and eeding habi s
(beha iou s) (monophagous, oligophagous, and polyphagous) o hese ec o s. Fo example,
No h Ame ican as e yellows phy oplasmas (16S I-A,-B) we e ansmi ed expe imen ally by
he polyphagous lea hoppe Mac os eles asci ons and o he ec o s o 191 plan species
belonging o 42 amilies (McCoy e al., 1989). No all ec o ing insec s can ansmi all
phy oplasmas and he e a e speci ic in e ac ions o a pa icula phy oplasma wi h i s insec
ec o . Some phy oplasmas, such as peach X-disease phy oplasma, may be ansmi ed by
se e al species o lea hoppe s; o he s, such as elm yellows phy oplasma, appea o be
ansmi ed by one o only a ew species (Lee and Da is, 1992).
1.5. Phylogene ic posi ion o phy oplasmas
Phy oplasmas ha e di e ged om g am-posi i e eubac e ia, and belong o he Genus
phy oplasma wi hin he Class Mollicu es and o de Acholeplasma ales (Figu e.1.5.). Cu en ly
he phy oplasma is a candida us s a us which is used o bac e ia ha canno be cul u ed.
Figu e.1.5. Phy oplasmas a e i micu es. A. Phylogene ic ela ionships o se e al bac e ial clades con aining
bac e ial pa hogens. B. The 5 phylogene ic g oups wi hin he Class Mollicu es. Plan pa hogenic/symbio ic
bac e ia a e indica ed in g een. GL, gene loss; WL, loss o cell wall. Figu e aken om Saskias phy oplasma
websi e, h p://www.jic.ac.uk/s a /saskia-hogenhou /index.h m.

In oduc ion
9
Recen ly, phylogene ic analyses based on 16S RNA and ibosomal p o ein gene sequences
ha e e ealed ha he uncul u ed phy oplasmas o m a la ge disc e e monophyle ic clade
wi hin he class mollicu es
(Gunde sen e al., 1994). Phy oplasma axonomic g oups a e based
on di e ences in he agmen sizes p oduced by he es ic ion diges o he 16S RNA gene
sequence (RFLP) o by compa ison o DNA sequences om 16S/23S space egions
(Hodge s e al., 2007).
1.6. Managemen and con ol o phy oplasma diseases
Me hods o con ol a y conside ably om one disease o ano he , depending on he kind o
pa hogen, he hos , he in e ac ion o he wo, and many o he a iables (Ag ios, 2004).
Mos se ious diseases o c op plan s appea on a ew plan s in an a ea yea a e yea , sp ead
apidly, and a e di icul o cu e a e hey ha e begun o de elop. The e o e, almos all
con ol me hods a e aimed a p o ec ing plan s om becoming diseased a he han a cu ing
hem a e hey ha e become diseased (Ag ios, 2004).
In con olling phy oplasmal diseases, he p ima y conce n is o en p e en ion a he han
ea men due he e is no known cu e o phy oplasmal in ec ions. Howe e , in ec ed plan s o
do man p opaga i e issue can be eed o phy oplasma by hea ea men .
1.6.1. P e en ion s a egy
P opaga e om seed o om phy oplasma- ee plan s, ha means selec p opaga ing ma e ial
om sou ces known o be ee o disease o indexed ee o disease.
Elimina e pe ennial and biennial weed hos s and e adica e known diseased ees as soon as
hey occu . The e o e, emo al o phy oplasma in ec ed plan s elimina es sou ces o in ec ion.
The e o e, ea ly, as , speci ic and sensi i e de ec ion and diagnosis o phy oplasmas a e e y
impo an o e ec i e p e en ion s a egies, especially because phy oplasmas may ha e a
e y long la ency pe iod. Howe e , he mos p omising s a egy o a oiding phy oplasma
disease is he iden i ica ion o de elopmen o esis an plan a ie ies (Welli e , 1999). In
o de o ad ance his ield o esea ch basic knowledge abou he epidemiology, he
pa hogenici y mechanisms o he phy oplasmas, he e ec s o en i onmen al ac o s on
disease and symp om de elopmen , and he na u e o esis ance/ ole ance in hos plan s is
equi ed
In oduc ion
10
1.6.2. Con ol o insec ec o s
“When he pa hogen is in oduced o sp ead by an insec ec o , con ol o he ec o is as
impo an as and some imes easie han, he con ol o he pa hogen i sel . In he case o
i uses, phy oplasmas, and as idious bac e ia, howe e , o which insec s a e he mos
impo an sp eading agen s, insec con ol has been help ul in con olling he sp ead o hei
diseases only when i has been ca ied ou in he a ea and on he plan s on which he insec s
o e win e o eed be o e hey en e he c op. Con olling such diseases by killing he insec
ec o s wi h insec icides a e hey ha e a i ed a he c op has a e p o ed su icien .
The e o e, in cases whe e he insec ec o is known and he ime o i s occu ence
es ablished, insec icide p og ams may be o alue when di ec ed a he ec o be o e i
becomes es ablished on he plan s. Typically, insec icide sp ays a e o limi ed alue since
mig a ing ec o s may ansmi he phy oplasma be o e he insec icide kills hose” (Ag ios,
2004).
1.6.3. Managemen s a egy
Because phy oplasmas lack a cell wall , hey a e esis an agains an ibio ics ha in e ac wi h
cell wall syn hesis like penicillin bu o he an ibio ics wi h an al e na i e modes o ac ion like
e acyclines can inhibi hei g ow h (bac e ios a ic o phy oplasmas). The e o e, emission
o he disease symp oms can be achie ed expe imen ally by injec ing he an ibio ic
e acycline bu wi hou con inuous use o his an ibio ic, disease symp oms will eappea
again
(Da ies e al., 1968). In addi ion, an ibio ic ea men is expensi e and ime-consuming.
The e o e, he bes s a egy is o apply an e icien elimina ion p og am.
As a conclusion, he only dynamic way o con ol phy oplasma in ec ion has been o p e en
he eme gence by gua an y ha clean plan ing ma e ial is used, o by ques o ind and/o
b eed a ie ies o c op plan s ha a e esis an o ole an o he phy oplasma/insec ec o .
1.7. Ana omy o phloem cells
Phloem cells conduc
soluble o ganic ma e ial made du ing pho osyn hesis in lea es o es o
he plan . They a e ali e a ma u i y and end o s ain g een (wi h he s ain as g een). Phloem
cells a e usually loca ed inside he xylem. The wo mos common cells in he phloem a e he
companion cells and sie e ube cells.
In oduc ion
11
The sie e- ube cells lack a nucleus, ha e e y ew acuoles, bu con ain o he o ganelles. The
sie e ube is an elonga ed ank o indi idual cells, called sie e- ube membe s, a anged end o
end. The endoplasmic e iculum is concen a ed a he la e al walls. Sie e- ube membe s a e
joined end o end o o m a ube ha conduc s soluble o ganic ood (pho osyn ha es) ma e ials
h oughou he plan . The end walls o hese cells ha e many small po es and a e called sie e
pla es and ha e enla ged plasmodesma a (Esau, 1965). Companion cells e ain hei nucleus
and con ol he adjacen sie e cells (Figu e.1.6).
Figu e.1.6. Diag am o he longi udinal iew o phloem cells. This image is om Pu es e al., (1992). Li e:
The Science o Biology, 4 h Edi ion.
1.8. Some phy oplasma diseases o suga cane
1.8.1. Suga cane yellow lea synd ome
Yellows diseases ha e been known since he ea ly 1900s. One such disease, as e yellows,
was i s epo ed in 1902. Be o e 1967, i s causal agen was hough by plan pa hologis s o
be o i al o igin because i could no be cul u ed in a i icial media.
Suga cane yellow lea synd ome (YLS), cha ac e ized by a yellowing o he mid ib and
lamina, (Figu e.1.7), was i s epo ed in he 1960s om Eas A ica (Roge s, 1969) and la e
om Hawaii (Schenck, 1990), Sou h A ica (C onje e al., 1998) and Cuba (A ocha
e al.,
1999). I is now widely dis ibu ed in mos suga cane g owing coun ies om all con inen s.
Losses om 30% o o e 60% o suscep ible a ie ies ha e been epo ed (Schenck e al.,
1997; Coms ock e al., 1994; A ocha e al., 2000). Symp oms o YLS ha e been a ibu ed o
In oduc ion
12
many causes, bo h bio ic and a bio ic, bu he bio ic causes a e associa ed wi h in ec ion by
lu eo i us o by phy oplasmas in Hawaii, B azil, Aus alia, Sou h A ica, Cuba, he USA and
Mau i ius.
Phy oplasmas ha e been consis en ly associa ed wi h YLS, bu la en in ec ions also occu
(Bailey e al., 1996; C onje e al., 1998; A ocha, 2000; Aljanabi e al., 2001).
Figu e.1.7. Suga cane yellow lea synd ome (YLS). Suga cane yellow lea synd ome is cha ac e ized by a
yellowing o he mid ib and lamina. Symp oms consis o yellowing lea es wi h a b igh yellow mid ib, o en
when he es o he lamina is s ill g een. This pic u e was aken om Komo e al., 2010.
1.8.2. Suga cane whi e lea and suga cane g assy shoo
Suga cane whi e lea (SCWL) and suga cane g assy shoo (SCGS) occu only in he sou h-
eas Asian egion and no in he o he suga cane g owing a eas o he wo ld. Bo h a e caused
by a single phy oplasma ype ha is a membe o he SCWL g oup and appea s o in ec only
suga cane. The mos cha ac e is ic symp oms o SCWL a e he p esence o lea es wi h o al
chlo osis, p oli e a ing ille s and p onounced s un ing. The lea es a e na owe and smalle
han hose o heal hy plan s (Figu e.1.8). SCWL is na u ally ansmi ed by he lea hoppe
Ma sumu a e ix hi oglyphicus o ganism Ma sumu a (Ma sumo o e al., 1968). Reco ds on
mechanically ansmission as well as on ansmission by aphids ha e no been con i med
(Rishi and Chen, 1989).
In oduc ion
13
Suga cane g assy shoo (SCGS) is one o he mos impo an diseases o suga cane in India. I
was i s obse ed in 1949 (Chona, 1958). SCGS has been eco ded in mos suga cane-
g owing a eas o India and is known o occu also in Thailand (Wongkaew e al., 1997;
Sdoodee e al., 1999). SCGS disease is cha ac e ized by he p oduc ion o a la ge numbe o
hin, slende , ad en i ious ille s om he base o he a ec ed s ools. This p o use g ow h
gi es ise o a dense o c owded bunch o ille s bea ing pale yellow o chlo o ic lea es which
emain hin, na ow, educed in size and ha e a so ex u e. The ec o (s) esponsible o he
na u al sp ead o SCGS ha e no been iden i ied. The e a e epo s on ansmission o SCGS
by h ee di e en species o aphids as well as by he ulgo id P ou is a moes a Wes woo
(Chona e al., 1960; Edison e al., 1976). Howe e , hese epo s ha e no been con i med
(Rishi and Chen, 1989).
Figu e.1.8. Suga cane whi e lea (SCWL). SCWL disease is caused by phy oplasma in Thailand. The mos
cha ac e is ic symp oms o SCWL a e he p esence o lea es wi h o al chlo osis, p oli e a ing ille s and
p onounced s un ing. The lea es a e na owe and smalle han hose o heal hy plan s. This pic u e was aken
om Komo e al., 2010.
1.9. De ec ion o suga cane phy oplasma in ec ions
Suga cane phy oplasma in ec ions can be de ec ed by mic oscopic examina ion o phloem
issue sec ions s ained wi h he DNA luo och ome4-6 diamidino-2-phenylindole (DAPI)
(Seemülle , 1976; Sa indu and Cla k, 1993). This p ocedu e is simple, apid and no much

In oduc ion
14
expensi e. Howe e i is limi ed when he phy oplasma popula ion is e y low and une enly
dis ibu ed among he plan hos o gans, as is o en ue o suga cane.
Fo de ec ion and iden i ica ion o suga cane phy oplasmas, he powe ul PCR echnology has
widely been employed in se e al labo a o ies. I o e s se e al ad an ages o e o he me hods
including e sa ili y, ela i e simplici y, speci ici y and high sensi i i y. P ime s ampli ying
RNA gene sequences p o ed mos sui able o PCR. I may be pe o med as one- ound PCR
o by eampli ying he DNA agmen s ob ained in he i s ampli ica ion using in e nal
p ime s (nes ed-PCR). Ve y o en in a ec ed suga canes he phy oplasma numbe s a e so low
ha in ec ions could be iden i ied only h ough he highly sensi i e nes ed PCR assay (T an-
Nguyen e al., 2000; Aljanabi e al., 2001).
1.10. Suga cane yellow lea synd ome in Hawaii
A no el suga cane disease was obse ed in Hawaiian suga cane plan a ions in he 1990s,
cha ac e ized by a yellowing o he lea mid ib, which was ollowed by s un ed lea ops and
yield decline (Schenck, 1990, Leh e e al., 2009). Simila symp oms we e epo ed sho ly
la e om plan a ions in B azil, mainland USA and Sou h A ica (Vega e al., 1997;
Coms ock e al., 1994; Bailey e al., 1996).
The disease was called Yellow lea synd ome (YLS) and classi ied in 2000 as a “disease o
unknown o igin” (Lockha and C onje, 2000). Resea ch on he na u e o he causal agen was
con o e sial among plan pa hologis s. A RNA- i us was isola ed om symp oma ic plan s
and named Suga cane yellow lea i us (SCYLV). I was p oposed as he causal agen o
YLS (Bo h e al., 1994; Vega e al., 1997). A su ey o YLS-diseased suga cane plan s in
A ica ailed o e eal a close co ela ion be ween SCYLV and symp oms, a be e co ela ion
was seen be ween he p esence o a phy oplasma in ec ion and symp om exp ession (C onje e
al., 1998). The phy oplasma-caused disease was called Lea yellows (LY) in con as o he
i us-caused disease, which is now called Yellow lea (YL).
The phy oplasma was named Suga cane yellow lea phy oplasma (SCYLP). I was ound in
suga cane om Aus alia, Sou h A ica, Cuba, India and Mau i ius (A ocha e al., 1999,
2005a; C onje e al., 1998; Aljanabi e al., 2001; Gau e al., 2008), some imes oge he wi h
SCYLV. Se e e suga cane diseases in Sou h-Eas Asia and A ica a e known o be caused by
phy oplasma (Ma cone, 2002), o example Whi e lea (Chen and Kusalwong, 2000), G assy
shoo (Viswana han, 2000), G een g assy shoo (Pliansinchai and P ammanee, 2000) and
In oduc ion
15
Ramu s un (Suma and Jones, 2000). Twen y- i e di e en phy oplasma isola es we e
ob ained om No h Aus alian suga cane plan s and none o hem was closely ela ed o
Whi e lea and G assy shoo , al hough also none o hem could be ela ed o suga cane disease
symp oms (T an-Nguyen e al., 2000).
Many publica ions deal wi h he Suga cane yellow lea i us and he associa ed disease, o
example i s wo ldwide dis ibu ion (Abu Ahmad e al., 2006; Komo e al., 2010), i s
nucleo ide sequence (Moonan e al., 2000; Smi h e al., 2000), he ansmission o he plan
(Schenck and Leh e , 2000; Leh e e al., 2007) and he physiological e ec s on he in ec ed
plan (Yan e al., 2009). The i us-caused lea yellowing synd ome is now accep ed as an
impo an , wo ldwide h ea o suga yield (G isham e al., 2002; Leh e e al., 2009). Also
he Sou h A ican suga indus y, o which o iginally he phy oplasmas we e hough o be
he main eason o YLS, appea s o be p edominan ly in ec ed by SCYLV and no by
phy oplasma (Ru he o d e al., 2004). Howe e , he YLS-p oblem is no ully sol ed ye .
The Hawaiian suga cane cul i a s we e di e en ia ed in o so-called suscep ible cul i a s
which con ain ela i ely high i es o SCYLV, and esis an cul i a s wi h 100 ime’s lowe
i us i es (Zhu e al., 2010). Expe imen s wi h in ec ed and i us- ee plan s o he same
cul i a indica ed ha he i al in ec ion led o highe symp om exp ession and o yield losses
(Leh e and Komo , 2008). The pic u e became less clea , when suscep ible cul i a s (i. e.
wi h high i us- i e) and esis an cul i a s (i. e. wi h low i us i e) we e compa ed. The
co ela ion be ween symp om exp ession and SCYLV-p esence was no s ic , some s ongly
in ec ed cul i a s exhibi ed ela i ely li le symp oms and some esis an cul i a s showed
symp oms, al hough a low in ensi y (Leh e and Komo , 2008). The e o e he ques ion a ose,
whe he some o he Hawaiian suga cane cul i a s we e also in ec ed by Suga cane yellow
lea phy oplasma (SCYLP), hus causing lea yellowing symp oms independen o o oge he
wi h SCYLV. The simul aneous p esence o SCYLV and SCYLP was epo ed o agg a a e
he lea yellowing symp om exp ession in suga cane (Aljanabi e al., 2001).
So a suga cane whi e lea o suga cane g assy shoo symp oms had no been epo ed in
Hawaiian plan a ions; howe e he p esence o a low-symp om pa hogen such as SCYLP may
ha e escaped a en ion o b eede s and g owe s. The e a e wo epo s abou phy oplasma
diseases in Hawaii, one abou wa e c ess yellows caused by an As e yellows ype
phy oplasma and ansmi ed by an acciden ally in oduced lea hoppe (Bo h e al., 2002;
2006), he o he abou a yellows disease o a na i e ee, Dodonaea iscosa, caused by a
Wes e n X-disease phy oplasma (Bo h e al., 1995).
In oduc ion
16
We es ed Hawaiian cul i a s (and o compa ison a ew cul i a s om Cuba, Egyp and
Sy ia) o phy oplasma o e eal whe he suga cane phy oplasma is a ound in Hawaii and in
Hawaiian suga cane plan a ions.
The main objec i es o his p ojec we e o de e mine he ollowing:
1. Possible associa ion o phy oplasma(s) in ec ion wi h YLS symp oms in suga cane plan s,
using o molecula echniques, namely PCR, o a mo e accu a e de e mina ion.
2. Which ype(s) o phy oplasma(s) a e associa ed wi h YLS symp oms in suga cane plan s
om Hawaii b eeding s a ion, Hawaii plan a ions, Cuba, Middle Eas and a eas in Thailand?
3. How does his ype(s) compa e o o he known phy oplasma ypes by phylogene ic
analysis?
4. Could i be ha Hawaiian plan a ions ha e phy oplasma a e ho wa e ea men ?
5. Can suga cane aphid (Melanaphis saccha i) ansmi he de ec ed phy oplasma o
suga cane plan s?
6. Ul as uc u al changes o he lea e ana omy and cy ology by phy oplasma in ec ion and
cy ological loca ion o phy oplasma.
Ma e ial and Me hods
17
2. Ma e ial and Me hods
2.1. Ma e ial
Balance (Me le P1210)
Cen i uges, Type cen i uge 5403 (Eppendo )
Cen i uge, Type Mik o 20 (He ich)
Cen i uge, Type UNIVERSAL 32R (He ich)
Diamond kni e ( ype 35°, Dia ome, Biel, Swi ze land)
Elec on mic oscope, Type ZEISS 902 (Zeiss, Obe kochem)
Gel Elec opho esis, Type GNA 100 (Pha macia LKB)
Gene powe supply, ype GPS 200/400 (Pha macia)
Mini-Ve ical Gel Elec opho esis, Type SE 250 and SE 260 (Migh y small II)
The momixe com o (Eppendo )
The mal cycle , Type PTC- 100 (MJ Resea ch)
The mal cycle , Type Mas e cycle pe sonal, wi h hea ed lid and 1 pe sonal ca d, 115 V/60
Hz (Eppendo )
The mal cycle , ype MyiQ qPCR de ec ion sys em o single-colou expe imen a ion (Bio-
ad)
Spec opho ome e , Type 650 (Beckman)
Ul a cu mic o ome (Leica Mic osys ems, We zla , Ge many)
Vo exe , Type REAX-1R (Heidolph)
Hea ed magne ic s i e , Type MR 82 (Heidolph)
Mic owa e o en, Type KOR- 6115 (Alaska)
Nanopho ome e , Type UV/Vis spec opho ome e (Implen)
pH-mV-me e , Type 531 (Knick)
UV-SYSTEME (NTAS)
2.1.2. Chemicals and Enzymes
2.1.2.1. Chemicals
Aga ose NEOO (Ca l Ro h GmbH)
30% Ac ylamide
10% Ammonium Pe sul a e
Ma e ial and Me hods
24
2.2.3. Polyme ase Chain Reac ion (PCR) o he de ec ion o phy oplasmas
Symp oma ology had been one he majo c i e ia o diagnosing he phy oplasma disease
be o e molecula -based me hods become a ailable. I emains he impo an clue used o
p elimina y iden i ica ion o pu a i e phy oplasmal diseases.
The polyme ase chain eac ion (PCR) is a apid p ocedu e o in i o enzyma ic ampli ica ion
o a speci ic segmen o DNA (Donald
e al., 2006)
. PCR has been used du ing he las yea s
o he de ec ion o la ge numbe o mic oo ganisms, also including phy oplasmas. Se e al
uni e sal p ime pai s designed o he ampli ica ion o he 16S RNA gene o phy oplasmas
we e es ed. The me hod ound o gi e consis en esul s was he nes ed PCR (Hein ich e al.,
2001; S i as a a e al., 2005).
2.2.3.1. De ini ion o Nes ed PCR
Nes ed PCR is a a ia ion o he polyme ase chain eac ion (PCR), in ha wo pai s (ins ead
o one pai ) o PCR p ime s a e used o ampli y a agmen . The i s pai o PCR p ime s
ampli ies a agmen simila o a s anda d PCR. Howe e , a second pai o p ime s called
nes ed p ime s (as hey lie) a e nes ed wi hin he i s agmen ) bind inside he i s PCR
p oduc agmen o allow ampli ica ion o a second PCR p oduc which is sho e han he
i s one (Pé ez de Rozas e al., 2008), (Figu e.2.1).

Ma e ial and Me hods
25
Figu e.2.1. A diag am illus a ing o he me hod o nes ed PCR. Figu e aken F om Wikipedia, he ee
encyclopaedia.
The ad an age o nes ed PCR is ha i he w ong PCR agmen was ampli ied, he
p obabili y is qui e low ha he egion would be ampli ied a second ime by he second se o
p ime s. Thus, nes ed PCR is a e y speci ic PCR ampli ica ion. Fu he mo e, he double
ampli ica ion in he nes ed-PCR inc eases he sensi i i y o PCR eac ion in 2-3 loga i hmic
uni s when compa ed wi h con en ional PCR (Lindq is , 1999; Ma silio e al., 2005).
2.2.3.2. Nes ed PCR Reac ion
Nes ed PCR equi es wo se s o p ime s which a e used o ampli y a speci ic DNA agmen
using wo sepa a e uns o PCR. A s anda d eac ion mix u e o 25 µl consis ed o he
ollowing:
Ma e ial and Me hods
26
10X Taq bu e
dNTP mix (200 µM each dNTP)
Fo wa d and e e se p ime s (0.4 µM)
Taq DNA polyme ase (5 U/µl)
Templa e DNA (100 ng)
dd H
2
O o inal olume 25 µl
2.2.3.3. Fi s ound o PCR
Nucleic acid samples we e dilu ed in s e ile dis illed wa e o gi e a inal concen a ion o 100
ng/ul and in some cases DNA concen a ions we e no adjus ed a e ex ac ion, bu used as
isola ed, 1 µl o DNA solu ion was used pe eac ion ube.
2.2.3.4. Nes ed ound o PCR
One mic o li e o dilu ed (1:30 o 1:20) PCR p oduc s om he i s ound was used as he
empla e in he second ampli ica ion. In mos cases i s PCR p oduc s we e used wi h any
dilu ion. The PCRs (30 cycles) we e done wi h an au oma ic he mal cycle in 25µl eac ion
ubes. Se e al uni e sal p ime pai s, which we e p e iously designed, based on he
phy oplasma RNA ope on, o he ampli ica ion o phy oplasmal DNA we e es ed
(Figu e.2.2 and Figu e.2.3). The me hod ound o gi e consis en esul s was he nes ed PCR.
Howe e , i was amazing when some p ime pai s didn’ wo k con inually and we had o es
in his case o he p ime pai s o check i he nega i e esul s we e alse due he p ime pai s
o due he phy oplasmas disappea ed om ou g eenhouse suga cane plan s. Since his
phenomenon may occu especially in ou g eenhouse when he e a e no insec ec o s o
phy oplasmas and he plan s ep oduce by ege a i e p opaga ion he e o e, he i e o
phy oplasma would be lowe gene a ion by gene a ion.
2.2.3.5. Nes ed-PCR assay (I)
The p ime pai combina ion used in he i s ound was P1/P7 while he nes ed p ime pai
was R16F2n/ R16R2 (Table.2.3).
Pa ame e s o he PCR assays using ex e nal p ime pai (P1/P7) we e: dena u a ion s ep a
94°C o 30 s (4 min o he i s cycle), annealing o 1.5 min a 55°C and p ime ex ension
o 1.5 min (10 min in inal cycle) a 72°C.
Ma e ial and Me hods
27
Pa ame e s o he PCR assays using in e nal (nes ed) p ime pai (R16F2n/R16R2) we e:
dena u a ion s ep a 94°C o 30 S (4 min o he i s cycle), annealing o 1.5 min a 56°C
and p ime ex ension o 1.5 min (10 min in inal cycle) a 72°C.
Table.2.3. Oligonucleo ide p ime s used o nes ed-PCR assay I.
P ime Loca ion Type o PCR
P1 (Fo wa d) 16S Fi s
P7 (Re e se) 23S Fi s
R16F2n (Fo wa d) 16S Nes ed
R16R2 (Re e se) 16S Nes ed
2.2.3.6. Nes ed-PCR assay (II)
The p ime pai s combina ions used in he i s and nes ed ounds o nes ed-PCR assay (II) a e
indica ed in (Table.2.4).
Pa ame e s o he PCR assays using ex e nal p ime pai SN910601/P6 we e: dena u a ion
s ep a 94°C o 30 s (4 min o he i s cycle), annealing o 1 min a 54°C and p ime
ex ension o 1.5 min (10 min in inal cycle) a 72°C.
Pa ame e s o PCR using in e nal p ime pai R16F2n/R16R2 ,which ampli ies 1250bp DNA
agmen , we e: dena u a ion s ep a 94°C o 30 s (4 min o he i s cycle), annealing o 1
min a 56°C, and p ime ex ension o 1.5 min (10 min in inal cycle) a 72°C.
Table.2.4. Oligonucleo ide p ime s used o nes ed-PCR assay II.
P ime Loca ion Type o PCR
SN910601 (Fo wa d) 16S Fi s
P6 (Re e se) 16S Fi s
R16F2n (Fo wa d) 16S Nes ed
R16R2 (Re e se) 16S Nes ed
2.2.3.7. Nes ed-PCR assay (III)
The p ime pai s combina ions used in he i s and nes ed ounds o nes ed-PCR assay (III)
a e indica ed in (Table.2.5).
Ma e ial and Me hods
28
The pa ame e s o he PCR assays using ex e nal p ime pai MLO-X/MLO-Y we e:
dena u a ion s ep a 94°C o 30 s (4 min o he i s cycle), annealing o 1 min a 58°C and
p ime ex ension o 1.5 min (10 min in inal cycle) a 72°C.
The pa ame e s o PCR using in e nal p ime pai P1/P2, which ampli ies 210 bp DNA
agmen , we e: dena u a ion s ep a 94°C o 30 s (4 min o he i s cycle), annealing o 45
s a 62°C, and p ime ex ension o 1 min (10 min in inal cycle) a 72°C.
Table.2.5. Oligonucleo ide p ime s used o PCR assay III.
P ime Loca ion Type o PCR
MLO-X (Fo wa d) 16S Fi s
MLO-Y (Re e se) space egion (nea 23S) Fi s
P1 (Fo wa d) 16S (nea he space egion) Nes ed
P2 (Re e se) " RNA-Ile" (nea he space
egion) Nes ed
2.2.3.8. Nes ed-PCR assay (IV)
The p ime pai s combina ions used in he i s and nes ed ounds o nes ed-PCR assay (IV)
a e indica ed in (Table.2.6).
Pa ame e s o he PCR assays using ex e nal p ime pai U-1/ MLO-7 we e: dena u a ion s ep
a 94°C o 30 s (4 min o he i s cycle), annealing o 1 min a 56°C and p ime ex ension
o 1.5 min (10 min in inal cycle) a 72°C.
Pa ame e s o PCR using in e nal p ime pai MLO-X/MLO-Y, which ampli ies 700bp DNA
agmen , we e: dena u a ion s ep a 94°C o 30 s (4 min o he i s cycle), annealing o 1
min a 60°C, and p ime ex ension o 1.5 min (10 min in inal cycle) a 72°C.
Table.2.6. Oligonucleo ide p ime s used o PCR assay IV.
P ime Loca ion Type o PCR
U-1 (Fo wa d) 16S Fi s
MLO-7 (Re e se) 23S Fi s
MLO-X (Fo wa d) 16S Nes ed
MLO-Y (Re e se) space egion (nea 23S) Nes ed
Ma e ial and Me hods
29
Figu e.2.2. Diag amma ic ep esen a ion o loca ion o used p ime pai s and expec ed size o hei
ampli ied p oduc s based on phy oplasma RNA ope on.
Figu e.2.3. Diag amma ic ep esen a ion o a phy oplasma RNA ope on and genomic loca ion o p ime s
used o phy oplasma de ec ion.
2.2.4. Aga ose Gel Elec opho esis
PCR p oduc s we e elec opho esed on 1% aga ose gel, s ained wi h e hidium b omide and
DNA bands isualized using a UV ansillumina o
Aga ose gel elec opho esis was used o isualize and isola e DNA molecules ollowing PCR
ampli ica ion. Aga ose (1%) was dissol ed in TBE bu e by hea ing in a mic owa e.
A e cooling, 1 µ o a 1 mg/ml e hidium b omide solu ion was added pe 50 ml gel and he
gel was pou ed. Gels we e un a 80-100 V o 1 hou .
PCR III
PCR IV
PCR II
PCR I

Ma e ial and Me hods
30
2.2.5. Diges ion o nes ed-PCR p oduc s
By RFLP analysis o PCR-ampli ied 16S RNA gene, he phy oplasmas de ec ed can be
di e en ia ed and classi ied (Lee e al., 1993). The basic echnique o de ec ing RFLPs
in ol es agmen ing he samples o DNA o (PCR p oduc s) by he es ic ion enzymes.
Res ic ion enzymes ecognize speci ic nucleo ide sequences and clea e DNA molecules a a
posi ion ei he wi hin o ou side hei ecogni ion si e (Robe s and Kenne h, 1976). These
enzymes a e impo an ools o nume ous applica ions, including es ic ion agmen leng h
polymo phism (RFLP) analysis o PCR p oduc s .The esul ing DNA agmen s a e hen
sepa a ed by hei leng h h ough gel elec opho esis.
RFLP analysis o PCR-ampli ied 16S RNA gene sequences wi h a numbe o es ic ion
enzymes was used by Lee e al., (1993) and Schneide e al., (1993) o di e en ia e a ious
phy oplasmas by hei dis inc RFLP pa e ns. This p ocedu e p o ed o be simple, eliable,
and p ac ical.
Ou nes ed PCR p oduc s we e analyzed by single enzyme diges ion wi h di e en es ic ion
endonucleases HpaIII, Hin I, KpnI, MesI and RsaI (MBI Fe men as).
The eac ion mix u e (30 µl) consis ed o he ollowing:
Reagen s:
10 µl PCR p oduc s
2 µl 10X ecommended bu e o es ic ion enzyme
1-2 µl (10-20 u) es ic ion enzymes
17 µl wa e nuclease ee
The eac ion mix u es we e incuba ed in he incuba o a 37°C o 3-16 h.
2.2.5.1. Inac i a ion o es ic ion enzymes
Inac i a ion o es ic ion enzymes ollowing a diges ion eac ion is o en equi ed o
downs eam applica ions. The mal inac i a ion is a con enien me hod used o e mina e
enzyme ac i i y. The majo i y o es ic ion enzymes can be hea -inac i a ed a 65°C o 80°C
in 20 min. Diges ed p oduc s we e sepa a ed by elec opho esis on 5% polyac ylamide gels.
Nex RFLP pa e ns we e compa ed wi h hose p e iously published.
Ma e ial and Me hods
31
2.2.6. Polyac ylamide Gel Elec opho esis
Nondena u ing polyac ylamide gels a e used o he sepa a ion and pu i ica ion o agmen s
o double-s anded DNA.
2.2.6.1. S eps o ope a ion
Assembling he appa a us and p epa ing he gel solu ion
Cas ing he gel
Loading he samples and unning he gel
2.2.6.2. Special equipmen
The SE 250 Migh y small II is a minia u e e ical slab gel uni in ended o apid
elec opho esis o nucleic acid samples o small olume.
2.2.6.3. De ec ion o DNA in polyac ylamide gels by s aining
Unlike aga ose gels, polyac ylamide gels canno be cas in he p esence o e hidium b omide
because he dye inhibi s polyme iza ion o he ac ylamide. Howe e , e hidium b omide can be
used o s ain he polyac ylamide gel a e elec opho esis. In o de o de ec o DNA he gels
we e gen ly subme ged in he app op ia e s aining solu ion. We used jus enough s aining
solu ion o co e he gel comple ely and he gels we e s ained o 30 min a oom
empe a u e.Then he gels we e emo ed om he s aining solu ion and placed on he UV
ansillumina o and pho og aphed.
2.2.7. Sequencing and phylogene ic analysis o ibosomal DNA
By di ec sequence analysis o RFLP analysis o PCR-ampli ied p oduc s, he phy oplasmas
de ec ed can be di e en ia ed and classi ied. Se e al classi ica ion sys ems ha e been
p oposed ei he di ec ly based on sequence analysis o indi ec ly, by RFLP analysis o PCR-
ampli ied 16S RNA gene.
In o de o ampli y he 16S/23S space egion we used P1/P7 o i s PCR and P4/P7 o
second PCR (Sma e al., 1996). P4/P7 PCR p oduc was pu i ied om aga ose gels using
Aga ose Gel DNA Ex ac ion Ki (Roche Diagnos ics GmbH). The DNA sample was
sequenced in one di ec ion using P4 p ime . Un o una ely, his p ime pai didn’ wo k well
Ma e ial and Me hods
32
o some cul i a s. The e o e, we used MLO-X/MLO-Y o i s PCR and P1/P2 o nes ed-
PCR o ampli y pa ially he 16S/23S space egion. In o de o ampli y he 16S RNA we
used R16F2n/R16R2 o nes ed PCR. Nes ed-PCR p oduc s we e cleaned up using High pu e
PCR p oduc s pu i ica ion Ki (Roche). DNA samples we e sequenced in bo h di ec ions
using nes ed p ime pai s.
2.2.7.1. Sample P epa a ion o Value Read Se ice in Tubes
The alue ead is he se ice o choice o as and eliable s anda d sequencing eac ions.
I is highly au oma ed o allow apid p ocessing o plasmids o PCR p oduc s. We used 1.5 ml
ubes (no addi ional sealing wi h Pa a ilm) o samples and p ime s and we used one ube pe
sequencing eac ion. The DNA samples (pu i ied PCR p oduc s) we e dissol ed in he elu ion
bu e (10 mM T is-HCl, pH 8.5) and he concen a ions o hese pu i ied PCR p oduc s we e
adjus ed o ge he inal concen a ion 10 ng/µl o 2 ng/µl in a minimum olume o 15 µl and
he equi ed p ime concen a ions we e 2 pmol/µl wi h minimum o al olume 15 µl. The
DNA samples (pu i ied PCR p oduc s) we e di ec -sequenced in an ABI 3730XL au oma ed
sequence using he sequencing se ice o Eu o ins MWG Ope on Ebe sbe g, Ge many
(h p://www.eu o insdna.com/
)
. Nex , sequences we e compa ed wi h o he s in GenBank
da abase using BLAST p og am.The sequence da a we e deposi ed in GenBank.
2.2.8. Ho wa e ea men
“When a pa hogen is excluded om he p opaga ing ma e ial (seed, ube s, bulbs, nu se y
s ock, g a s, and cu ings) o hos , i is o en possible o g ow he hos ee o ha pa hogen
o he es o i s li e” (Ag ios, 2004).Vege a i e p opaga ing ma e ial ee o pa hogens ha
a e sys emically dis ibu ed h oughou he plan s ( i uses, i oids, and phy oplasmas) is
ob ained om mo he plan s ha had been es ed and shown o be ee o he pa icula
pa hogen o pa hogens. Fu he mo e, he new plan s mus be g own in pa hogen- and ec o -
ee soil and hen be p o ec ed om ai bo ne ec o s.
Phy oplasma may be ansmi ed by he p opaga ion o scions and o cu ing collec ed om
diseased plan . The e o e, in ege a i ely p opaga ed c ops like suga cane; phy oplasma can
be eadily sp ead o new loca ions h ough in ec ed s em cu ing i sui able p ecau ions a e no
aken. These p ecau ions include cold- and ho wa e ea men and issue cul u e (Pa messu
e al., 2002).
Ma e ial and Me hods
33
A ho wa e ea men is an e ec i e me hod o he con ol o numbe o plan s pes s and
diseases (plan pa hogens) including phy oplasmas.
Ho wa e ea men (HWT) has been p oposed since 1966 by Caudwell a 30°C o 72 h in
o de o cu e do man woody plan ma e ial om phy oplasmas. A e wa d o he wo ks
showed he e ec i eness o he ea men agains hese pa hogens (Lhe minie e al., 1990;
Tassa -Subi a s e al., 2003). Howe e , (HWT) mus be ca e ully applied because may
in e e e wi h he i ali y o plan ma e ial. Thus, (HWT) o do man canes o plan s aims a
phy oplasma elimina ion wi hou any al e a ion in hei ege a i e de elopmen capaci y. In
addi ion he ea men demons a es a posi i e e ec o sani a ion agains se e al bac e ial
diseases, pes s and insec s (including eggs) which may be p esen on plan ma e ial.
2.2.8.1. P epa a ion o he plan ma e ial p io o he ho wa e ea men
In ec ed plan s o do man p opaga i e o gans can be o ally eed o phy oplasmas by hea
ea men . In ec ed plan s a e kep in g ow h chambe s a 30°C o 37°C o se e al days,
weeks, o mon hs; bu do man o gans a e imme sed in ho wa e (Ag ios, 2004).
Soaking induces a he mal shock suscep ible o modi ying he physiological s a e o he plan
ma e ial (b eaking o bud do mancy, inducing s o age losses). The e o e, in o de o p e en a
poo ege a i e de elopmen , he plan ma e ial should be he mally p epa ed o he ea men
by s o age o 12 o 48 hou s a oom empe a u e in a humid and ae a ed chambe .
Fu he mo e, he empe a u e a e imme sion and he ea men du a ion should be espec ed
and a e ea men , he plan ma e ial should be le o se back o oom empe a u e (a oid
di ec con ac wi h cold wa e ).
2.2.9. Suga cane aphid ansmission es
2.2.9.1. Insec ea ing
Melanaphis Saccha i (Suga cane aphid) insec s we e p o ided om Hawaii Island.
Colonies o Melanaphis Saccha i we e es ablished on phy oplasma-in ec ed suga cane plan s.
2.2.9.2. Plan ma e ial
All es plan s aised om single-eye se s ha had ecei ed cold- and ho -wa e ea men o
ho -wa e ea men we e nega i e (phy oplasma ee) when es ed by nPCR p io o being
Resul s
40
In ano he expe imen , DNA o he posi i e con ol was dilu ed by inc easing quan i ies o
DNA which had been ex ac ed om suga cane lea es o plan s which we e known o be
phy oplasma- ee. The pu pose was o es , whe he compounds om suga cane lea es may
possibly inhibi he ampli ica ion o phy oplasma 16S RNA gene. The suga cane ex ac by
i sel did no gi e an amplicon. The posi i e con ol sample always yielded a posi i e signal,
e en when dilu ed up o 40- old by suga cane DNA, he 50- old dilu ion wi h suga cane DNA
did no gi e an amplicon anymo e (Figu e.3.1).
Figu e.3.1. Nes ed PCR-p oduc s o posi i e and nega i e con ols and o a posi i e con ol, which was
mixed wi h inc easing amoun s o suga cane DNA. The posi i e con ol (pos. c.) was a sample o Ame ican
as e yellows phy oplasma g own in pe iwinkle (ob ained om D . Be accini, Bologna). The wa e con ol 1+2
was wi h wa e ins ead o DNA in he i s PCR ound and u he ampli ied in he second PCR ound. Wa e
con ol 2 con ained wa e ins ead o i s ound amplicon. The suga cane DNA was om Egyp ian cul i a
(
Ph-
8013)
which had been shown o be phy oplasma- ee (suga cane c.) using he p ime pai s combina ion (P1/P7
and R16F2n/R16R2). The ma ke (M) was DNA GeneRule 100 bp plus (MBI Fe men as). The a ows poin o
he phy oplasma-speci ic band o 1.2 kbp.
Though PCR analysis is ou ine echnique o phy oplasma de ec ion, i ’s s ill mee ing some
di icul ies, a leas wi h some p ime s: se e al p ime pai s and hei combina ion a e
ecommended (Hein ich e al., 2001). In ou lab, PCR assay was ca ied ou wi h di e en
p ime pai s combina ion. To ampli y egion ha includes he 16S RNA gene, he space
egion and he s a o 23S RNA gene o he phy oplasma genome.
The e o e, each suga cane

Resul s
41
sample was in es iga ed o phy oplasma by using ou nes ed-PCR assays which we e
numbe ing as ollowing: (I), (II), (III) and (IV). The p ime pai s and hei sequences which
used in each assay we e men ioned a ma e ial and me hods chap e .
3.1.2. Sou ces o suga cane samples
Suga cane samples, which we e in es iga ed in ou lab, we e ob ained om di e en a eas
and di e en da es. Some o hem we e ob ained as s em cu ings and g own in he
g eenhouse while o he s we e ha es ed and conse ed as ai -d ied lea es. Mos o hem a e
showing suga cane yellow lea synd ome symp oms whe eas o he s we e symp omless
(Table.3.1).
Table.3.1. O iginal sou ces o suga cane samples. Mos o suga cane samples we e ob ained om Hawaiian
Islands while o he s om Thailand. In addi ion, some suga cane samples we e aken om Cuba and Middle Eas
a ea including Egyp and Sy ia. Some o hem we e ob ained as s em cu ings and g own in he g eenhouse
whe eas o he s we e collec ed and conse ed as ai -d ied lea es.
O iginal sou ce Loca ion Da e o ge ing
suga cane samples
Type o suga cane
sample
Hawaii B eeding s a ion o
HARC in Maunawili 2003 S em cu ings
Hawaii Plan a ions
(Maui and Kauai) 2009 Sun-d ied lea es
Hawaii Fo me plan a ion
ields (Maui, Kauai
and Hawaii)
2009 Sun-d ied lea es
Hawaii B eeding s a ion o
HARC in Maunawili 2010 Sun-d ied lea es
Hawaii Close o o me
plan a ion ields in
Hawaii
2011 Sun-d ied lea es
Hawaii B eeding s a ion o
HARC in Maunawili 2011 Sun-d ied lea es
Hawaii plan a ion
(Maui) 2011 Sun-d ied lea es
Thailand Fa me ields
(Bang Ph a) 2010 Sun-d ied lea es
Thailand B eeding s a ion
(Khon Kean) 2010 Sun-d ied lea es
Thailand Fa me ields
(Suphan Bu i) 2011 Sun-d ied lea es
Cuba B eeding s a ion 2005 S em cu ings
Egyp B eeding s a ion 2008 S em cu ings
Sy ia Fa me ields
(Baniyas) 2008 S em cu ings
Resul s
42
3.2. Phy oplasma in suga cane in Hawaii, Cuba, Egyp and Sy ia
Six suga cane cul i a s om Hawaii we e ob ained in 2003 as s em cu ings om he
b eeding s a ion o HARC in Maunawili, Oahu , h ee SCYLV-suscep ible cul i a s (H87-
4094, H73-6110, H65-7052) and h ee SCYLV- esis an cul i a s (H78-7750, H78-4153,
H87-4319).
These s em cu ings we e g own in g eenhouse o uni e si y o Bay eu h. In addi ion,
cul i a s om Cuba we e ob ained om D . O ega, Habana, in 2005 also as s em cu ings
and g own beside Hawaiian samples. Cul i a s om Egyp we e ob ained as s em cu ings
om he Uni e si y o Gizah in 2008. The cul i a om Sy ia was ob ained as s em cu ings
in 2008 om a a me ´s ield nea Baniyas. The ques ion was as he ollowing:
A e hese
ob ained suga cane samples in ec ed wi h phy oplasma?
3.2.1. Phy oplasma de ec ion by nes ed-PCR assay (I) and iden i ica ion by RFLP
DNA was ex ac ed om sou ce lea es and es ed o phy oplasma by nes ed-PCR assay (I)
wi h p ime pai s (P1/P7 and R16F2n/R16R2) in 2008. All cul i a s con ained phy oplasma
showing an amplicon a 1.2 kbp, al hough appa en ly a di e en i es, o example H73-
6110, a s ongly SCYLV-in ec ed cul i a , had only a low SCYLP- i e (Figu e.3.2). The
Cuban cul i a s (C10-5173, CP43-62, JA60-5) and one cul i a om Egyp (G84-47) was
also in ec ed by phy oplasma, al hough appa en ly a a low i e, no howe e he cul i a G -
954 and Ph-8013 om Egyp and he plan om Sy ia (Figu e.3.3). The esul s wi h c . G -
954, Ph-8013 and he Sy ian cul i a hus we e an impo an nega i e con ol, showing ha
he e is no DNA sequence in he suga cane genome which gi es a alse posi i e signal wi h
his p ime pai .
Resul s
43
Figu e.3.2. Phy oplasma in Hawaiian and Cuban suga cane cul i a s. DNA p epa ed om lea es o he
indica ed cul i a s was es ed wi h p ime s P1/P7 and R16F2n/R16R2. The posi i e con ol (pos. c1) was
phy oplasma as e yellows om pe iwinkle ob ained om D . Seemülle , Dossenheim, pos.c2 was phy oplasma
as e yellows om pe iwinkle ob ained om D . Be accini, Bologna. The wa e con ol 1+2 was wi h wa e
ins ead o DNA in he i s PCR ound and u he ampli ied in he second PCR ound. The ma ke M1 is DNA
ladde Fas Rule Middle ange (MBI Fe men as, agmen sizes: 4, 2, 1, 0.5 kbp).The a ows poin o he
phy oplasma-speci ic band o 1.2 kbp.
Figu e.3.3. Phy oplasma in Egyp ian and Sy ian suga cane cul i a s. DNA p epa ed om lea es o he
indica ed cul i a s was es ed wi h p ime s P1/P7 and R16F2n/R16R2. Re-ampli ica ion o aliquo o i s PCR
wa e con ol wi h nes ed p ime combina ion is in lane W. The ma ke M2 DNA GeneRule 1kb (MBI
Fe men as). The a ows poin o he phy oplasma-speci ic band o 1.2 kbp.
G84
-
47
G
-
954
Ph
-
8013
Sy ian c .
M2
W
Resul s
44
Res ic ion agmen analysis had been success ully applied o di e en ia e be ween he
phy oplasma s ains (Ki kpa ick e al., 1994; Lee e al., 1998; Valiunas e al., 2007).
The amplicons o he second ound o PCR (I) we e subjec ed o h ee es ic ion enzymes
which we e diagnos ic o he phy oplasma s ains. The ob ained RFLP pa e ns we e
compa ed wi h hose p e iously published by Lee e al., 1998. The es ic ion pa e ns
iden i ied he phy oplasma om Hawaiian cul i a s and om one Cuban cul i a as belonging
o he As e yellows phy oplasma “Ca. Phy oplasma as e is”, whe eas he phy oplasma om
he Cuban cul i a CP4362, which o iginally had been b ed in Canal Poin , Flo ida and om
JA605, belonged o he Wes e n X-disease phy oplasma “Ca. Phy oplasma p uni”. Howe e ,
a second p o ile was clea ly isible in he gel in some Hawaiian suga cane cul i a s ha
indica e o he possible p esence o phy oplasmas ela ed o ice yellow dwa g oup (16S XI),
“
Ca. Phy oplasma o yzae” (Figu e.3.4 and Table.3.2).
a
Resul s
45
Figu e.3.4. Res ic ion agmen analysis o PCR p oduc s om Hawaiian and Cuban suga cane cul i a s
con aining phy oplasma. The nes ed-PCR p oduc s we e ampli ied wi h p ime s R16F2n/R16R2 ollowing
diges ion wi h RsaI (a), HpaII (b) o KpnI (c) and sepa a ed on 5% polyac ylamide. The posi i e con ols (As e
yellows and Wes e n X-disease) we e used as e e ences. The black a ows indica e o second p o ile which
e eals possible p esence o ice yellow dwa (16S XI) phy oplasmas as mixed in ec ion .The ma ke (M) is
Mass Rule DNA Ladde , low ange (MBI Fe men as), agmen sizes 1031, 900, 800, 700, 600, 500, 400, 300,
200, 100, and 80 bp.
b
c

Resul s
46
Table.3.2. Resul s o nes ed-PCR assay (I) and iden i ica ion o phy oplasmas based on RFLP analyses.
Two phy oplasmas we e iden i ied in mixed in ec ion in some Hawaiian suga cane cul i a s: one ela ed o as e
yellows g oup (16S I) while he o he en a i ely ela ed o ice yellow dwa g oup (16S XI). +, phy oplasma
de ec ed.
Suga cane a ie ies O iginal sou ce De ec ion o
phy oplasma in 2008
based on PCR assay
(I)
Phy oplasma g oup
based on RFLP
analyses o 16S RNA
gene
H87- 40 94 Hawaii + As e yellows
C10- 51 73 Cuba + As e yellows
H78- 77 50 Hawaii + As e yellows and ice
yellow dwa
Cp- 43 62 Flo ida + X-disease
H78- 41 53 Hawaii + As e yellows and ice
yellow dwa
H73- 61 10 Hawaii + As e yellows and ice
yellow dwa
H65- 70 52 Hawaii + As e yellows and ice
yellow dwa
JA- 60 5 Cuba + X-disease
H87- 43 19 Hawaii + As e yellows and ice
yellow dwa
H87- 40 94 VF Hawaii + As e yellows
3.2.2. Phy oplasma de ec ion by nes ed-PCR assay (II) and iden i ica ion by RFLP
Oligonucleo ide p ime s used o nes ed-PCR assay (II) we e (SN910601/P6) o i s -PCR
and (R16F2n/R16R2) o nes ed-PCR. Hawaiian, Egyp ian and Sy ian suga cane samples
g own in g eenhouse we e es ed by his PCR assay. Acco ding o his analyse ou Hawaiian
cul i a s, wo Egyp ian cul i a s and Sy ian cul i a we e nega i e o phy oplasma
(Figu e.3.5, Table.3.3 and 3.4).
Resul s
47
Figu e.3.5. Nes ed-PCR assay (II) p oduc s (1.2kb) ampli ied wi h p ime s (SN910601/P6,
R16F2n/R16R2). (a): Hawaiian suga cane samples g own in g eenhouse. (b): Egyp ian and Sy ian suga cane
samples. The ma ke M was GeneRule 100 bp plus (MBI Fe men as). The a ows poin o he phy oplasma-
speci ic band o 1.2 kb. Acco ding o his analysis wo Hawaiian suga cane cul i a s H78-4153 and H87-4319
and one Egyp ian cul i a G8447 we e posi i e o phy oplasmas.
P oduc s o nes ed-PCR assay (II) we e analyzed by
RFLP analysis using single enzyme
diges ion wi h es ic ion endonucleases (HpaII and MseI). The ob ained RFLP pa e ns we e
compa ed wi h hose p e iously published by Lee e al., 1998. Acco ding o his diges ion he
Hawaiian cul i a s H78-4153 and H87-4319 con ain phy oplasmas all in as e yellows g oup
(Figu e.3.6; a and Table.3.3) whe eas Egyp ian cul i a
G8447 in ec ed wi h phy oplasma
belongs o ice yellow dwa g oup (Figu e.3.6; b and Table.3.4). Howe e , u he RFLP
analysis is equi ed o di e en ia e i his phy oplasma belongs o suga cane whi e lea s ain
(SCWL) o suga cane g assy shoo one (SCGS).
1,2kb
G8447
M
Ph8013
GT549
Sy ia
1,2kb
4153
7052
4319
6110
7750
4094
M
a
b
b
a
Resul s
48
3.2.3. Phy oplasma de ec ion by nes ed-PCR assay (III)
Oligonucleo ide p ime s used o nes ed-PCR assay III we e (MLO-X/MLO-Y) o i s -PCR
and (P1/P2) o nes ed-PCR. Acco ding o his analyse all Hawaiian cul i a s, one Egyp ian
(G8447) and Sy ian cul i a we e posi i e o he p esence o phy oplasma bu no he o he
wo Egyp ian cul i a s (Figu e.3.7, Table.3.3 and 3.4).
MseI
MseI
HpaII
H87
-
4319
H87
-
4319
H78
-
4153
H78
-
4153
HpaI
I
M
M
M
G8447
G8447
HpaI
I
MseI
M
M
a
b
a
a
b
Figu e.3.6. RFLP p o iles o nes ed-PCR assay (II)
p oduc s. These p oduc s ampli ied wi h p ime pai
(SN910601/P6, R16F2n/R16R2) o Hawaiian suga cane
samples (a) and Egyp ian suga cane samples (b) g own in
g eenhouse ollowing single enzyme diges ion wi h (HpaII
and MseI) and sepa a ion on 2% aga ose gel. The ma ke M
was GeneRule 100 bp plus (MBI Fe men as). Acco ding o
his diges ion he Hawaiian cul i a s H78-4153 and H87-
4319 con ain phy oplasmas all in as e yellows g oup while
in Egyp ian cul i a G8447 alls in ice yellow dwa g oup.
Resul s
49
Figu e.3.7. Nes ed-PCR assay (III) p oduc s (0.2kb) ampli ied wi h p ime pai (MLO-X/MLO-Y, P1/P2).
(a): Hawaiian suga cane samples. (b): Egyp ian suga cane samples. (c): Sy ian suga cane sample g own in
g eenhouse. Re-ampli ica ion o aliquo s o i s PCR wa e con ols wi h nes ed p ime combina ion a e in lanes
W. The ma ke M was GeneRule 100 bp plus (MBI Fe men as).The a ows poin o he phy oplasma-speci ic
band o 0. 2 kb.
3.2.4. Phy oplasma de ec ion by nes ed-PCR assay (IV) and iden i ica ion by RFLP
Oligonucleo ide p ime s used o nes ed-PCR assay IV we e (U-1/MLO-7) o i s -PCR and
(MLO-X/MLO-Y) o nes ed-PCR. Acco ding o hese da a he p ime s used in his assay
could no de ec phy oplasmal DNA p esen in all Hawaiian cul i a s which we e posi i e o
phy oplasma as men ioned abo e (Table.3.3). Phy oplasmal DNA in Sy ian and one Egyp ian
cul i a (G8447) was de ec ed wi h his p ime pai (Figu e.3.8 and Table.3.4)
210bp
4153
7052
4319
6110
7750
4094
0,2kb
G844
7
0,2kb
Sy ian
M
M
Ph801
G
549
W
W
W
W
a
b
c
M
W
W
Resul s
56
al. 2010) and hey may ha e become in ec ed by phy oplasma in case ha he app op ia e
insec ec o s we e p esen . Samples o he uppe mos ully un olded lea es o plan s ound in
o me plan a ion ields we e collec ed, sun-d ied and es ed o phy oplasma.
3.4.1. Phy oplasma de ec ion and iden i ica ion
The ex ac ed DNA om hese samples ga e ampli ica ion only wi h p ime pai s o PCR
assay (III). The ob ained amplicons we e sequenced in o de o iden i y he phy oplasma
(Figu e.3.13 and Table.3.6).
Figu e.3.13. phy oplasma in o me Hawaiian plan a ions suga cane samples (2009). Nes ed-PCR assay
(III) p oduc s (0,2kb) ampli ied wi h p ime s (MLO-X/MLO-Y, P1/P2). The samples M10 o M13 we e om
Maui plan a ion, H1 o H4 we e om Hawaii plan a ion, K2 o K5 om Kauai, all as ai -d ied and hen o en-
d ied. Re-ampli ica ion o aliquo s o i s PCR wa e con ols wi h nes ed p ime combina ions a e in lanes W.
The ma ke (M) was DNA GeneRule 100 bp plus (MBI Fe men as).
M1
0
H1
H2
H3
H4
K
2
K3
K4
K5
210 bp
M
M
W
W
W
W
M1
3
M1
2
M1
1
0.2kb

Resul s
57
Table.3.6.
Phy oplasma in suga cane samples om o me Hawaiian plan a ions (2009). Resul s o
phy oplasma de ec ion based on nes ed-PCR assays and iden i ica ion based on DNA sequencing o nes ed-PCR
assay (III) using P1/P2. +, phy oplasma de ec ed; -, phy oplasma no de ec ed. The samples (H1 o H4) we e
om Hawaii while he samples (M10 o M13) we e om Maui and he samples (K2 o K5) we e om Kauai.
Island and
collec ion si e phy oplasma de ec ion
based on PCR assay
(I), (II) and (IV)
phy oplasma de ec ion
based on PCR assay
(III)
Phy oplasma g oup
based on DNA
sequencing o PCR
assay (III)
(P1/P2)
Hawaii
H1 - + As e yellows
H2 - + As e yellows
H3 - + As e yellows
H4 - + As e yellows
Maui (H65-7052)
M10 - +
M11 - + Rice yellow dwa
M12 - + Rice yellow dwa
M13 - + Rice yellow dwa
Kauai (H65-7052)
K2 - + As e yellows
K3 - + As e yellows
K4 - +
K5 - +
3.5. Phy oplasma in suga cane in Hawaiian b eeding s a ion (2010)
Six suga cane cul i a s om Hawaiian b eeding s a ion o HARC in Maunawili, Oahu we e
sen om D . Zhu in 2010 as sun-d ied lea es in o de o es o phy oplasmas.
3.5.1. Phy oplasma de ec ion and iden i ica ion
Only he p ime s o PCR assay (III) ampli ied DNA in hese suga cane cul i a s. Howe e ,
he ob ained bands we e e y weak, he e o e; only wo o hem we e sequenced (Figu e.3.14
and Table.3.7).
0,2kb
4153
7052
4319
6110
7750
4094
M
W
Figu e.3.14. phy oplasma in Hawaiian
b eeding s a ion suga cane samples (2010).
Nes ed-PCR assay (III) p oduc s (0.2kb)
ampli ied wi h p ime s (MLO-X/MLO-Y,
P1/P2) ob ained om Hawaiian suga cane
b eeding s a ion (2010) as ai -d ied samples. Re-
ampli ica ion o aliquo o i s PCR wa e
con ol wi h nes ed p ime combina ion is in
lane W. The ma ke (M) was DNA GeneRule
100 bp plus (MBI Fe men as).
Resul s
58
Table.3.7. Phy oplasma in suga cane plan s om Hawaiian (Maunawili, HARC) b eeding s a ion (2010).
Resul s o phy oplasma de ec ion based on nes ed-PCR assays and iden i ica ion by DNA sequencing o nes ed-
PCR assay (III) using P1/P2. +, phy oplasma de ec ed; -, phy oplasma no de ec ed.
Sample name phy oplasma
de ec ion based on
n-PCR assay
(I)& (II)& (IV)
phy oplasma
de ec ion based on
n-PCR assay
(III)
Phy oplasma
g oup based on
DNA sequencing
(P1/P2)
H65-7052 - + Rice yellow dwa
H73-6110 - + As e yellows
H78-7750 - +
H78-4153 - +
H87-4094 - +
H87-4319 - +
3.6. Phy oplasma in suga cane in Hawaiian b eeding s a ion and plan a ions (2011)
In Feb ua y 2011, suga cane lea samples we e ha es ed om di e en a eas in Hawaiian
Islands including plan a ion HC&S, Maui and Maunawili b eeding s a ion. Then, hese
suga cane samples we e sun-d ied in o de o es he p esence o phy oplasm in ou lab. Mos
o hese samples we e aken om suga cane plan s a e showing suga cane yellow lea
synd ome symp oms (Figu e.3.15).
3.6.1. Phy oplasma in suga cane in Hawaiian plan a ion
These plan a ions ob ained he cul i a s om he b eeding s a ion o HARC in Maunawili,
Oahu, ollowed by se e al cycles o ield es ing and mul iplica ion. The ques ion was i
phy oplasmas can be esponsible o YLS in his plan a ion ield and i he e is signi ican
co ela ion be ween he p esence o phy oplasma and showing suga cane yellow lea
synd ome since some samples we e s ongly o sligh ly symp oma ic while o he s
asymp oma ic. Samples om uppe mos ully un olded sou ce lea es om plan s o di e en
cul i a s we e collec ed in he plan a ion ields and sun-d ied. The samples om Maui we e
om HC&S plan a ion ( ields 702, 500, 608 and 809, all sou h-eas o Puunene). In each case
3 lea samples om 3 di e en plan s we e es ed.
Resul s
59
3.6.1.1. Phy oplasma de ec ion and iden i ica ion
DNA was ex ac ed om sou ce lea es and each suga cane sample was in es iga ed o he
p esence o phy oplasma by using ou nes ed-PCR assays (I), (II), (III) and (IV)
wi h
di e en p ime pai combina ions as was cla i ied be o e. Only nes ed-PCR assay (III) ga e
posi i e eac ions. Ou esul s showed ha symp oma ic
and non- symp oma ic plan s con ain
phy oplasma ((Figu e.3.16 and Table.3.8).
A
B
C
Figu e.3.15. Suga cane lea es
showing symp oms o in ec ion
wi h yellow lea synd ome (A) and
(B), compa ed wi h an unin ec ed
g een lea (C). Suga cane yellow
lea synd ome symp oms a e caused
by se e al agen s including
phy oplasma.
Resul s
60
Table.3.8.
Phy oplasma in suga cane plan s om Hawaiian HC&S plan a ion o Maui island, close o
Puunene (2011). Resul s o phy oplasma de ec ion based on nes ed-PCR assays and iden i ica ion based on
DNA sequencing o p oduc s o nes ed-PCR assay (III) using P1/P2. Acco ding o he DNA sequencing analysis
he suga cane samples con ain ice yellow dwa phy oplasma. +, phy oplasma de ec ed; -, phy oplasma no
de ec ed.
O de o
Suga cane
a ie ies
Suga cane
cul i a and Lea
condi ion
phy oplasma
de ec ion based
on
PCR assay
(I), (II), (IV)
phy oplasma
de ec ion based
on
PCR assay
(III)
Phy oplasma
g oup based on
DNA
sequencing
(P1/P2)
1 H65-7052,
6 mon hs
Non-
symp oma ic
- +
2 H65-7052,
6 mon hs
symp oma ic
- - -
3 H73-3567,
4 mon hs
Non-
symp oma ic
- - -
4 H87-4319,
9 mon hs
Non-
symp oma ic
- + Rice yellow
dwa
5 H87-4319,
9 mon hs
sligh ly-
symp oma ic
- +
6 H86-3792,
6 mon hs
Non-
symp oma ic
- + Rice yellow
dwa
7 H87-5794,
9 mon hs
Non-
symp oma ic
- + Rice yellow
dwa
210bp
1
2
3
4
5
6
7
M
Figu e.3.16. Phy oplasma in Hawaiian
HC&S plan a ion o Maui island, close o
Puunene (2011). Nes ed-PCR assay (III)
p oduc s (0.2kb) ampli ied wi h p ime s
(MLO-X/MLO-Y, P1/P2) o suga cane
samples ob ained om Hawaiian plan a ion
HC&S, Maui, close o Puunene; (2011) as
sun-d ied lea es. The ma ke (M) was DNA
GeneRule 100 bp plus (MBI Fe men as).
Resul s
61
3.6.2. Phy oplasma in suga cane in Hawaiian b eeding s a ion (2011)
Samples om uppe mos ully un olded sou ce lea es om 10 cul i a s o suga cane plan s
we e collec ed om HARC b eeding s a ion in Maunawili ( ields A, B and P which a e
widely dis an om each o he ) and sun-d ied o phy oplasma in es ga ion. Names o hese
suga cane cul i a s a e indica ed in (Table.3.9).
These cul i a s we e p e iously es ed o
suga cane yellow lea i us (SCYLV) by Leh e e al., 2001.The esul s o his es a e
indica ed in (Table.3.11).
3.6.2.1. Phy oplasma de ec ion by nes ed-PCR assay (II) and iden i ica ion
DNA was ex ac ed and es ed o phy oplasma by nes ed-PCR assay (II). Mos o hese
suga cane cul i a s p oduced an amplicon a 1.2 kbp, al hough appa en ly a di e en i es
(Figu e.3.17). P oduc s o nes ed-PCR assay (II) we e diges ed wi h es ic ion endonucleases
(HpaII and MseI). The ob ained RFLP pa e ns we e compa ed wi h hose p e iously
published by Lee e al., 1998. Acco ding o his diges ion hese Hawaiian suga cane samples
con ain phy oplasmas all in ice yellow dwa g oup (Figu e.3.18 and Table.3.9). Howe e ,
u he RFLP analysis is equi ed o di e en ia e i his phy oplasma belongs o suga cane
whi e lea s ain (SCWL) o suga cane g assy shoo one (SCGS) as men ioned be o e.

Resul s
62
Table.3.9. Phy oplasma in Hawaiian (Maunawili, HARC) b eeding s a ion suga cane samples (2011).
Resul s o phy oplasma de ec ion based on nes ed-PCR assay (II) and iden i ica ion based on RFLP analysis
using single enzyme diges ion wi h HpaII and MseI. Sample numbe s indica e o he o de o samples in nex
igu es (3.17) and (3.18). +, phy oplasma de ec ed; -, phy oplasma no de ec ed.
Samples numbe Samples name phy oplasma de ec ion
based on
PCR assay
(II)
Phy oplasma g oup
based on RFLP
wi h HpaII and MseI
1 H87-4094 ield A11 +
2 H78-3567 “ + Rice yellow dwa
3 H87-4319 “ +
4 H65-7052 “ +
5 H50-7209 “ + Rice yellow dwa
6 H78-4153 “ + Rice yellow dwa
7 H77-4643 “ + Rice yellow dwa
8 H73-6110, ield A22 +
9 H32-8560, ield B31
10 H78-7750, ield B62 +
11 H87-4319 “ + Rice yellow dwa
12 H78-3606 “ - -
13 H77-4643, ield
P11a +
14 H78-3606, ield P11 - -
15 H50-7209, ield P12 - -
16 H87-4319 “ +
17 H65-7052 “ - -
18 H78-7750 “ +
19 H73-6110, ield P13 +
Resul s
63
Figu e.3.17. Phy oplasma de ec ion in suga cane samples om Hawaiian (Maunawili, HARC) b eeding
s a ion (2011). Nes ed-PCR assay (II) p oduc s (1.2kb) ampli ied wi h p ime s (SN910601/P6, R16F2n/R16R2).
Samples numbe s and names a e indica ed in able (3.9) abo e. Re-ampli ica ion o aliquo s o i s PCR wa e
con ols wi h nes ed p ime combina ions a e in lanes W. The ma ke (M) was DNA GeneRule 100 bp plus
(MBI Fe men as).
Figu e.3.18. Phy oplasma iden i ica ion in suga cane samples om Hawaiian (Maunawili, HARC)
b eeding s a ion (2011). RFLP p o iles o nes ed-PCR assay (II) p oduc s (1.2kb) ampli ied wi h p ime s
(SN910601/P6, R16F2n/R16R2) ollowing single enzyme diges ion wi h HpaII (a) and MseI (b). The ma ke M
was GeneRule 100 bp plus (MBI Fe men as).
3.6.2.2. Phy oplasma de ec ion by nes ed-PCR assay (III) and (IV) and iden i ica ion
Acco ding o PCR assay (III) esul s mos o hese suga cane cul i a s we e posi i e o
phy oplasma (Figu e.3.19). This is also ue o PCR assay (IV) (Figu e.3.20). P oduc s o
PCR assay (IV) we e diges ed wi h es ic ion endonuclease (Hin I). Acco ding o his
diges ion hese Hawaiian suga cane samples con ain phy oplasma s ain o suga cane whi e
lea (SCWL) (Figu e.3.21 and Table.3.10).
1,2kb
3567
7209
4153
4643
6110
M
M
4319
1
2
3
4
5
6
7
10 8
11 12 13 14 15 16 17 18 19 W
W
W
W
3567
M
7209
4153
4643
a
b
Resul s
64
Table.3.10. Phy oplasma in suga cane samples om Hawaiian (Maunawili, HARC) b eeding s a ion
(2011). Resul s o phy oplasma de ec ion based on nes ed-PCR assays (III) and (IV) and iden i ica ion based on
RFLP analysis o (IV) p oduc s using single enzyme diges ion wi h (Hin I). Sample numbe s indica e o he
o de o samples in nex igu es (3.19 and 3.20). +, phy oplasma de ec ed; -, phy oplasma no de ec ed.
Sample
numbe
Sample
name Phy oplasma
de ec ion based on
PCR assay
(III)
Phy oplasma
de ec ion based on
PCR assay
(IV)
Phy oplasma
s ain based on
RFLP wi h
(Hin I)
1 H87-4094
ield A11 + + Suga cane whi e
lea
2 H78-3567 + + “
3 H87-4319 + + “
4 H65-7052 + + “
5 H50-7209 + + “
6 H78-4153 + + “
7 H77-4643 - + “
8 H73-6110
ield A22 + + “
9 H32-8560
ield B31 - - -
10 H78-7750
ield B62 + + “
11 H87-4319 - - -
12 H78-3606 + + “
13 H77-4643
ield P11a + + “
14 H78-3606
ield P11 + + “
15 H50-7209
ield P12 - + -
16 H87-4319 + - “
17 H65-7052 - + -
18 H78-7750 + + “
19 H73-6110 + + “
Resul s
65
Figu e.3.19. Phy oplasma de ec ion in suga cane samples om Hawaiian (Maunawili, HARC) b eeding
s a ion suga cane samples (2011). Nes ed-PCR assay (III) p oduc s (0.2kb) ampli ied wi h p ime s (MLO-
X/MLO-Y, P1/P2). Samples numbe s and names a e indica ed in (Table.3.10). Re-ampli ica ion o aliquo s o
i s PCR wa e con ols wi h nes ed p ime combina ion is in lanes W. The ma ke (M) was DNA GeneRule
100 bp plus (MBI Fe men as).
Figu e.3.20. Phy oplasma de ec ion in suga cane samples om Hawaiian (Maunawili, HARC) b eeding
s a ion (2011). Nes ed-PCR assay (IV) p oduc s (0.7kb) ampli ied wi h p ime s (U-1/MLO-7, MLO-X/MLO-Y).
Samples numbe s and names a e indica ed in (Table.3.10). Re-ampli ica ion o aliquo s o i s PCR wa e
con ols wi h nes ed p ime combina ion is in lanes W. The ma ke (M) was DNA GeneRule 100 bp plus (MBI
Fe men as).
0,7kb
210bp
1
2
3
4
5
6
7
10 9
8
11 12 13 14 15 16 17 18 19 W M W
1
2
3
4
5
6
7
10 8
11 12 13 14 15 16 17 18 19 W
M W
9
Resul s
72
Table.3.14. Phy oplasmas in suga cane samples
om p o inces o Bang Ph a and Khon Kean in Thailand
in 2010; based on nes ed-PCR assays. Samples (F1 o F8) we e om a me ields in p o ince o Bang Ph a
while he samples (S1 o S13) we e om b eeding s a ion also in p o ince o Bang Ph a. Samples (KK1 o
KK38) we e om p o ince o
Khon Kean.
Suga cane
sample Desiease
Symp oms PCR assay
(III) PCR assay
(IV) Phy oplasma
s ains based on
RFLP wi h (Hin I)
F1 Whi e ly + + Suga cane whi e
lea
F2 Lea spo + + “
F3 us + + “
F4 Yellow spo + + “
F5 Mosaic i us + + “
F6 Cu ly spindle + + “
F7 S un ed lea + + “
F8 Whi e lea + + “
S1 Spo ed mosaic - -
S2 Spo ed mosaic + + Unknown
S3 - -
S4 us - + Unknown
S5 us - + Unknown
S6 - -
S7 - -
S8 - -
S9 + -
S10 S eak mosaic + + Suga cane whi e
lea
S11 S eak mosaic + + Unknown
S12 S eak mosaic - -
S13 S eak mosaic - -
KK1 + + Suga cane whi e
lea
KK2 Mosaic + + “
KK3 G assy shoo + + “
KK4 Yellow mid ib + + “
KK11 Yellow mid ib + -
KK12 Lea scalel + + “
KK13 E ian hus c oss
yellow mid ib + -
KK14 mosaic + + “
KK17 mosaic + + “
KK18 E ian hus + -
KK20 mosaic + + “
KK21 Spo - -
KK32 whi e lea + + “
KK33 whi e lea + + “
KK34 whi e lea + + “
KK35 S ipe + + “
KK36 S ipe + + “
KK37 Yellow mid ib + + “
KK38 Yellow mid ib + + “

Resul s
73
Figu e.3.25; a, b and c. Phy oplasma de ec ion in Thia suga cane samples om Bang Ph a and Khon
Kean. Nes ed-PCR assay (III) p oduc s (0.2kb) o suga cane samples ob ained om a me ields (F1 o F8) and
b eeding s a ion (S9 o S11) in Bang Ph a and Khon Kean (KK1 o KK38) as sun-d ied lea es. Samples numbe s
and names a e indica ed in (Table 3.14). Re-ampli ica ion o aliquo s o i s PCR wa e con ols wi h nes ed
p ime combina ions a e in lanes W. The ma ke (M) was DNA GeneRule 100 bp plus (MBI Fe men as).
210 bp
F1
F2
F3
F7
F6
F5
F4
F8
M
W
W
210 bp
Kk1
2
4
3
11
12
13
14
17
M
Kk18
20
32
35
36
37
38
S9
S10
S11
M
W
W
0,2kb
a
b
c
Resul s
74
Figu e.3.26; a, b, c and d. Phy oplasma de ec ion in Thia suga cane samples om Bang Ph a and Khon
Kean. Nes ed-PCR assay (IV) p oduc s (0.7kb) o suga cane samples ob ained om a me ields (F1 o F8) and
b eeding s a ion (S10 o S11) in Bang Ph a and Khon Kean (KK1 o KK38) as sun-d ied lea es. Samples
numbe s and names a e indica ed in (Table 3.14). Re-ampli ica ion o aliquo s o i s PCR wa e con ols wi h
nes ed p ime combina ion is in lanes W. The ma ke (M) was DNA GeneRule 100 bp plus (MBI Fe men as).
0,7kb
F1
F2
F3
F4
F5
F6
F7
F8
W
M
kk1
3
4
11
12
14
17
20
18
32
35
M
W
W
36
38
S10
S11
M
M
W
a
b
c
d
0,7kb
2
13
Resul s
75
Figu e.3.27; a, b, c and d. Phy oplasma iden i ica ion in Thia suga cane samples om Bang Ph a and
Khon Kean. RFLP p o iles o nes ed-PCR assay (IV) p oduc s (0.7kb) o suga cane samples ob ained om
a me ields (F3 o F8) and b eeding s a ion (S2 o S11) in Bang Ph a and Khon Kean (KK1 o KK35) as sun-
d ied lea es ollowing single enzyme diges ion wi h (Hin I). The ma ke M was GeneRule 100 bp plus (MBI
Fe men as).
3.7.2. Phy oplasma in suga cane in Suphan Bu i p o ince (2011)
Suga cane samples we e sen om a me ields in p o ince o Suphan Bu i as sun-d ied
lea es. Fou samples we e aken om suga cane plan s showing yellow lea synd ome
symp oms and one sample was aken om plan shows suga cane g assy shoo symp oms.
3.7.2.1. Phy oplasma de ec ion and iden i ica ion
The ex ac ed DNA om hese samples ga e ampli ica ion only wi h p ime pai s o PCR
assay (III). Some p oduc s o his assay we e sequenced o he iden i ica ion (Figu e.3.28 and
Table.3.15).
F4 F3 F5 F7 F6 F8 M
M
M
Kk1
Kk3
Kk14
Kk32
Kk35
M
S10
S11
Kk2
Kk20
S2
S4
S5
M
M
a
b
c
d
0.5 kb
0.2 kb
0.5 kb
0.2 kb
0.5 kb
0.2 kb
Resul s
76
Table.3.15. Ou lines o phy oplasmas in Thai suga cane samples om Suphan Bu i.
Suga cane sample phy oplasma
de ec ion based on
PCR assay
(III)
phy oplasma
de ec ion based on
PCR assay
(IV)
Phy oplasma g oup
based on DNA
sequencing
(P1/P2)
1 + - Rice yellow dwa
2 + - „
3 + - "
4 + - "
G aasy shoo + - "
3.8. Es ablishmen o TaqMan qPCR assay as ano he es o phy oplasma
Mos uni e sal as well as speci ic phy oplasma diagnos ic p o ocols ely on nes ed PCR,
which, al hough ex emely sensi i e, is also ime-consuming and possess isks in e ms o
ca y-o e con amina ion be ween he wo ounds o ampli ica ion (Wein aub and Jones,
2010). Recen ly, di ec qPCR has eplaced he adi ional PCR in e o s o inc ease he speed
and sensi i i y o de ec ion and o imp o e echniques o mass sc eening (Wein aub and
Jones, 2010).
3.8.1. Pe o mance cha ac e is ics o qPCR
Pe o mance cha ac e is ics which include e iciency, limi o de ec ion and sensi i i y o
amplicons we e de e mined by ampli ying h ee sepa a ely p epa ed se s o dilu ion se ies o
h ee s anda d samples in wa e which include 1- phy oplasma-in ec ed pe iwinkle
1
2
3
4
M
M
W
G assy
shoo
0,2kb
a
b
Figu e.3.28; a and b. Phy oplasma in Thia
suga cane samples om Suphan Bu i.
Nes ed-PCR assay (III) p oduc s (0.2kb)) o
suga cane samples ob ained om a me
ields in Suphan Bu i as sun-d ied lea es.
Samples numbe s and names a e indica ed in
(Table.3.15). Re-ampli ica ion o aliquo o
i s PCR wa e con ol wi h nes ed p ime
combina ion is in lane W. The ma ke (M)
was DNA GeneRule 100 bp plus (MBI
Fe men as).
Resul s
77
(phy oplasmal DNA) 2- phy oplasma-in ec ed suga cane (phy oplasmal DNA) 3-
phy oplasma- ee suga cane (plan DNA). Since he copy numbe o a ge genes in he
s anda d samples is unknown, he s anda d cu es a e help ul o he e alua ion o PCR
e iciency and sensi i i y, bu no o absolu e quan i ica ion.
3.8.1.1. E iciency Measu emen
In his s udy, e iciency (E) alues we e measu ed using he C slope me hod. This me hod
in ol es gene a ing a dilu ion se ies o he a ge empla e and de e mining he C alue o
each dilu ion. A plo o C e sus log DNA concen a ion is cons uc ed (Figu es.3.29, 30 and
31). Ampli ica ion e iciency was calcula ed om he slope o his g aph using he equa ion:
Ex = 10^ (-1/slope) – 1. The e ec o e iciency is exponen ially dependen on cycle numbe .
I E=1, amplicon quan i y is duplica ed e e y cycle. I E=0.8 amplicon quan i y is only
duplica ed e e y 1. 2 cycle. The squa ed eg ession coe icien a e he linea eg ession (R²)
was also de e mined (Table.3.16 and 17).

Resul s
78
Figu e.3.29. A: S anda d cu e. S anda d cu e de e mined a six concen a ion le els ( anging om 10^0 o
10^-5) using 10- old dilu ion se ies o he e e ence sample (phy oplasma-in ec ed pe iwinkle). The h eshold
numbe s o PCR cycles (CT alue; means o iplica es) a e plo ed agains he dilu ion (log scale).B: Log- iew
o s anda d cu e cha . Th eshold; is an a bi a y le el o luo escence chosen on he basis o he baseline
a iabili y. C ; is de ined as he ac ional PCR cycle numbe a which he epo e luo escence is g ea e han
he h eshold.∆Rn; is an inc emen o luo escen signal a each ime poin . The ∆Rn alues a e plo ed e sus
he cycle numbe .
15
17
19
21
23
25
27
29
31
33
35
-4 -3 -2 -1 0 1 2
Th eshold Cycle
Log s a ing quan i y
A
B
Ampli ica ion cha
C
Th eshold
∆ Rn
S anda d cu e o qPCR
Resul s
79
Figu e.3.30. A: S anda d cu e.
S anda d cu e de e mined a six concen a ion le els ( anging om 10^0 o
10^-5) using 10- old dilu ion se ies o he e e ence sample (phy oplasma-in ec ed suga cane). The h eshold
numbe s o PCR cycles (CT alue; means o iplica es) a e plo ed agains he dilu ion (log scale). B: Log- iew
o s anda d cu e cha . ∆Rn; is an inc emen o luo escen signal a each ime poin . The ∆Rn alues a e
plo ed e sus he cycle numbe
12
14
16
18
20
22
24
26
28
30
32
-3 -2 -1 0 1 2 3
Th eshold Cycle
log s a ing quan i y
A
B
Ampli ica ion cha
∆ Rn
S anda d cu e o qPCR
Resul s
80
Figu e.3.31. A: S anda d cu e. S anda d cu e de e mined a six concen a ion le els ( anging om 10^0 o
10^-5) using 10- old dilu ion se ies o he phy oplasma- ee suga cane plan sample. The h eshold numbe s o
PCR cycles (CT alue; means o iplica es) a e plo ed agains he dilu ion (log scale). B: Log- iew o
s anda d cu e cha . ∆Rn; is an inc emen o luo escen signal a each ime poin . The ∆Rn alues a e plo ed
e sus he cycle numbe
8
10
12
14
16
18
20
22
24
26
28
-4 -3 -2 -1 0 1 2
Th esholed Cycle
Log s a ing quan i y
A
B
Ampli ica ion cha
∆ Rn
S anda d cu e o qPCR
Resul s
81
3.8.1.2. A i icial samples o es sensi i i y o qPCR assay
Pe o mance cha ac e is ics we e also e alua ed o he se ial dilu ion o phy oplasma-
in ec ed suga cane (phy oplasmal DNA) mixed wi h suga cane DNA, ins ead o wa e ,
isola ed om phy oplasma- ee suga cane lea es o imi a e eal in ec ed suga cane samples.
Thus, PCR sensi i i y was e alua ed o po en ial e ec s o hos -ma e ial inhibi ion. This un-
in ec ed suga cane ma e ial had al eady been es ed and con i med o be phy oplasma- ee
suga cane. A i icial samples imi a ing in ec ed suga cane samples we e p epa ed by se ial
dilu ions o phy oplasma-in ec ed suga cane DNA mixed wi h phy oplasma- ee suga cane
DNA. A plo o C e sus log DNA concen a ion is also cons uc ed as abo e
(
Figu e.3.32).
Resul s
88
The C alues di e ed conside ably among samples in he phy oplasma assay, while C alues
ob ained in he plan assay we e di e en sligh ly. This esul indica ed ha phy oplasma i e
was a iable.
3.8.3. Dis ibu ion o phy oplasma in suga cane
Q-PCR assay o phy oplasma 16S DNA was used o de e mine he dis ibu ion o he
phy oplasma wi hin in ec ed suga cane plan . The ela i e dis ibu ion o suga cane whi e lea
phy oplasma in di e en pa s o he plan was quan i ied using he compa a i e C me hod.
Th ee lea samples o phy oplasma in ec ed suga cane including whi e, a iega ed and g een;
and oo samples we e analysed
(
Figu e.3.33)
.
The phy oplasma was de ec ed in all es ed
o gans including lea es and oo s. I seems he e is co ela ion be ween i e o phy oplasma
and symp oms exp ession whe e he i e o phy oplasma in whi e lea was highe han
a iega ed and g een lea es. Lowe C alues co espond o highe ini ial quan i ies o
phy oplasma DNA empla e
(Table.3.28).
Table.3.28. Q-PCR esul s o he dis ibu ion o he phy oplasma in suga cane plan . CT is a h eshold
cycle numbe o qPCR assay. +,phy oplasma de ec ed.
Suga cane sample CT mean alue
(16S)
Phy oplasma
CT mean alue
(18S)
Suga cane
Phy oplasma
de ec ion
Whi e lea 11.56 08.31 +
Va iega ed lea 12.97 08.76 +
G een lea 14.02 08.79 +
Roo 12.28 08. 82 +

Resul s
89
Figu e.3.33. A: Thai suga cane plan in ec ed wi h suga cane whi e lea phy oplasma whe e some lea es a e
o ally bleaching whe eas o he s a e a iega ed and some g een lea es also exis s. This pic u e was aken h ee
mon hs pos ge mina ion compa ison wi h non-in ec ed suga cane plan s in (B).
3.9. Phylogene ic analysis o he phy oplasma s ains in suga cane
Res ic ion agmen analysis had been success ully applied o di e en ia e be ween he
phy oplasma s ains (Ki kpa ick e al., 1994; Lee e al., 1998; Valiunas e al., 2007). The
p oduc s o he second ound PCR we e subjec ed o es ic ion enzymes which we e
diagnos ic o he phy oplasma s ains. The es ic ion pa e ns iden i ied he phy oplasma
om Hawaiian cul i a s and om one Cuban cul i a as belonging o he As e yellows
phy oplasma, whe eas he phy oplasma om he Cuban cul i a CP4362, which o iginally had
been b ed in Canal Poin , Flo ida and om JA605, belonged o he Wes e n X-disease
phy oplasma (Figu e.3.4 and Table. 3.2). This classi ica ion was suppo ed by sequence
compa ison.
The comple e sequence o R16F2n/R16R2-ampli ied agmen s was de e mined o h ee
di e en suga cane cul i a s which a e in ec ed by h ee di e en phy oplasma isola es, wo
cul i a s a e om Cuba and one om Egyp (Figu e.3.34). The comple e sequence o
16S/23S in e genic space egion was de e mined o one Hawaiian suga cane cul i a using
he p ime pai P4/P7 (Figu e.3.34). The pa ial sequence o 16S/23S in e genic space egion
was also de e mined o o he wo Hawaiian suga cane cul i a and o one cul i a om
Thailand using he p ime pai P1/P2 (Figu e.3.34).
A
B
Resul s
90
Figu e.3.34. Diag amma ic ep esen a ion o genomic loca ion o p ime s used o DNA sequencing.
The ob ained nucleo ide sequences we e compa ed wi h sequences o phy oplasmas and
acholeplasmas om GenBank using he BLASTN p og am. Mul iple alignmen s o nea - ull-
leng h 16S RNA gene sequences om 22 phy oplasma and one Acholeplasma species and
mul iple alignmen s o 16S/23S in e genic space egion om 13 phy oplasma and wo
Acholeplasma species we e examined using MUSCLE so wa e. Phylogene ic ees o bo h
sequence pa s we e cons uc ed o e eal he posi ion o he isola ed phy oplasma s ains
om Hawaiian, Cuban, Egyp ian and Thai suga cane, ela i e o phy oplasma s ains which
had been isola ed om suga cane and o he plan s. Figu e.3.35 p esen s he wo phylogene ic
ees ha we e cons uc ed by maximum likelihood es ima ion wi h geneious p og am h ough
he PhyML so wa e (Guindon and Gascuel, 2003). Boo s ap analysis was pe o med 1.000
imes o e alua e b anch suppo s in a sound s a is ical amewo k.
The phy oplasma isola e (HM804282) om Cuban suga cane cul i a Ja605 clus e ed
oge he wi h o he s ains o X-disease g oup, among hem al eady epo ed suga cane
yellows phy oplasma s ain ound in Sou h A ica (AF056095) wi h a boo s ap alue o 48.7
and sha ed 99% sequence iden i y (Figu e.3.35 and
Table.3.29). O he Cuban suga cane
cul i a C10-5173 was in ec ed wi h phy oplasma s ain (HQ116553) clus e ed o he as e
yellows g oup, closely oge he wi h suga cane yellows phy oplasma om B azil (EU423900)
and maize bushy s un phy oplasma om Colombia (HQ530152) wi h a boo s ap alue o
49.6 and sha ed 99% sequence iden i y (Figu e.3.35 and Table.3.29).
Egyp ian suga cane cul i a G8447 con ains phy oplasma s ain (JN223446) clus e ed o he
ice yellow dwa g oup, closely oge he wi h so ghum g assy shoo phy oplasma om
Aus alia (AF509324) wi h a boo s ap alue o 81.1 and sha ed 99% sequence iden i y
(Figu e.3.35
and Table.3.29).
Resul s
91
The phylogene ic ee o he 16S/23S space egion con ained less phy oplasma en ies in
GenBank. The Hawaiian suga cane phy oplasma isola e (HQ116554) om cul i a H84-4094
and ano he Hawaiian suga cane phy oplasma isola e (JN223447) om unknown cul i a
ob ained om Hawaiian o me plan a ions as a di e en o iginal sou ce clus e ed o he as e
yellows g oup, closely oge he wi h wa e c ess yellows om Hawaii (AY665676) and
Russian po a o pu ple op phy oplasma (EU333399) wi h a boo s ap alue o 69.6 and sha ed
99% sequence iden i y (Figu e.3.35 and Table.3.29).
Hawaiian suga cane phy oplasma isola e (JN223448) om cul i a H78-7750
which ob ained
om Hawaiian b eeding s a ion o HARC in Maunawili, Oahu clus e ed o he ice yellow
dwa g oup, closely oge he wi h suga cane whi e lea phy oplasma om Taiwan
(AY139874) wi h a boo s ap alue o 86.6 and sha ed 98% sequence iden i y (Figu e.3.35
and Table.3.29).
Thai suga cane phy oplasma isola e (HQ917068) om unknown suga cane cul i a ob ained
om p o ince o Khon kaen clus e ed o he ice yellow dwa g oup, closely oge he wi h
suga cane whi e lea phy oplasma om Myanma (AB646271) wi h a boo s ap alue o 64.2
and sha ed 100% sequence iden i y (Figu e.3.35 and Table.3.29).
Resul s
92
Table.3.29. Phy oplasma s ains and hei GenBank accession numbe s used in his s udy o he
phylogene ic ees (Figu e.3.35). 16S RNA gene and 16S/23S in e genic space egion sequences o
phy oplasmas de e mined in his s udy a e in bold. Phy oplasma s ains o monoco yledonous plan s and s ains
which showed close sequence simila i y o he Hawaiian, Cuban, Egyp ian and Thai suga cane phy oplasma
we e selec ed o cons uc ion o he ees. The sequences om Acholeplasma axan hum and Acholeplasma
palmae we e used as ou g oups.
Phylogene ic ee o 16S RNA (a)
Accession numbe Phy oplasma s ain G oup
AF056095 Suga cane yellows phy oplasma ype I
(Sou h A ica) X-disease
AF411592 E ige on wi ches'-b oom phy oplasma Ash yellows
AF509324 So ghum g assy shoo phy oplasma
a ian I (Aus alia) Rice Yellow Dwa
AF498307 Coconu le hal yellowing phy oplasma Coconu le hal
yellowing
AJ550984 Be muda g ass whi e lea phy oplasma
(Sou he n I aly) Be muda whi e lea
AM261831 Suga cane g assy shoo phy oplasma
(India) Rice Yellow Dwa
AY197652 Spa ium wi ches'-b oom phy oplasma Apple p oli e a ion
AY736374 Napie g ass s un phy oplasma
(Kenya)
Rice Yellow Dwa
EF413055 So ghum e icilli lo um phy oplasma
(Mau i ius) X-disease
EF413056 Suga cane yellows phy oplasma clone
SC245 (Mau i ius) X-disease
EU294011 Malaysia Be muda g ass whi e lea
phy oplasma Be muda whi e lea
EU423900 Suga cane yellows phy oplasma ype I
(B asil) As e yellows
FM208260 Suga cane whi e lea (Thailand) Rice Yellow Dwa
GQ336993 Kidney bean li le lea phy oplasma
clone Z16 16S Peanu WB
GQ850122 Coconu oo wil phy oplasma isola e
RD3 (India) Rice Yellow Dwa
GU565959 Candida us Phy oplasma py i isola e
932801 Apple p oli e a ion
HM804282 Suga cane Ja60-5 yellow lea (Cuba) X-disease
HQ116553 Suga cane C1051-73 yellow lea
(Cuba) As e yellows
HQ530152 Maize bushy s un phy oplasma s ain
MBSColombia (Colombia) As e yellows
HQ589200 Milkweed yellows phy oplasma s ain
MWI(USA) X-disease
JF508514 Sesame phyllody phy oplasma s ain
Seph2 Peanu WB
JN223446 Suga cane g assy shoo phy oplasma
(Egyp ) Rice Yellow Dwa
NR_029152 Acholeplasma palmae s ain J233 Ou g oup
Resul s
93
Phylogene ic ee o 16S/23S in e genic space egion
(b)
Accession numbe Phy oplasma s ain G oup
AB243298 Suga cane g assy shoo phy oplasma
(India)
Rice Yellow Dwa
AB646271 Suga cane whi e lea phy oplasma
(Myanma )
Rice Yellow Dwa
AF434989 Texas Phoenix palm phy oplasma Coconu le hal
yellowing
AY139874 Suga cane whi e lea phy oplasma
(Taiwan) Rice Yellow Dwa
AY665676 As e yellows phy oplasma
“Wa e c ess” (Hawaii) As e yellows
DQ004923 Acholeplasma palmae Ou g oup
DQ400425 Acholeplasma axan hum Ou g oup
EU294011 Malaysia Be muda g ass whi e lea
phy oplasma Be muda whi e lea
EU333399 Russian po a o pu ple op
phy oplasma (Russia)
As e yellows
FN562932 Candida us Phy oplasma i is Elm yellows
HQ116554 Hawaiian suga cane H87-4094
yellow lea phy oplasma As e yellows
HQ589192 'Psammo e ix cephalo es' lowe
s un phy oplasma Rice Yellow Dwa
HQ917068 Suga cane whi e lea phy oplasma
(Thailand) Rice Yellow Dwa
JN223447 Hawaiian suga cane Phy oplasma As e yellows
JN223448 Suga cane whi e lea phy oplasma
(Hawaii) Rice Yellow Dwa

Resul s
94
a
Rice yellow dwa
X-disease
As e yellows
Resul s
95
Figu e.3.35.
Posi ion o he phy oplasma s ains om Hawaiian, Cuban, Egyp ian and Thai suga cane in
a phylogene ic ee oge he wi h o he phy oplasma isola es (Table.3.29). a: Phylogene ic ee cons uc ed
using 16S RNA sequences om 22 phy oplasma and one Acholeplasma species, b: Phylogene ic ee
cons uc ed using 16S/23S space sequences om 13 phy oplasma and wo Acholeplasma species. Ba
ep esen s phylogene ic dis ance o 2%. Numbe s on b anches a e con idence pe cen age ob ained om 1.000
boo s ap eplica es.
b
Rice yellow dwa
As e yellows
Resul s
96
3.10. Ho wa e ea men in o de o ge phy oplasma ee suga cane plan
Ho wa e ea men had been p oposed as a cu e o phy oplasma in do man woody plan
ma e ial (Edison and Ramak ishnan, 1972; Caudwell e al., 1997), because phy oplasmas ha e
only limi ed hea ole ance.
T ea men o in ec ed suga cane s alks wi h mode a ely high empe a u es such as 50°C o 2
h we e epo ed o success ully elimina e g assy shoo disease and whi e lea phy oplasma
om s em ma e ial. Ho wa e ea men o s em cu ings oge he wi h imme sion in a
ungicide solu ion is a ou ine p ac ice in Hawaiian suga cane plan a ions o p e en ungal o
o plan ed seed pieces. The ques ion was which empe a u e egimes and which incuba ion
du a ions a e needed o elimina e SCYLP om suga cane s ems and whe he he ou ine ho
wa e - ea men agains ungi had unin en ionally also cu ed om phy oplasma.
One-eye s em cu ings we e imme sed in ho wa e o de ined empe a u e and o de ined
pe iod, hen plan ed in s e ile soil in po s and kep in insec - igh mesh cages o ge mina ion
and g ow h. Indeed, incuba ion o seed pieces a 50°C o 30 min o longe was su icien o
elimina e phy oplasma (Table.3.30), i espec i e whe he i was om As e yellows o om
Wes e n X-disease ype. The incuba ion in ho wa e o 3 h had a de imen al e ec on seed
piece iabili y unless he ho wa e ea men was p eceded by 10°C incuba ion o 48h, a
p ocedu e ou inely used in he Aus alian and Cuban suga indus y.
3.10.1. Ho wa e ea men acco ding o Aus alian ecipe
Two suga cane cul i a s (H65-70 52 and H78-77 50) we e used as ma e ial in he ho wa e
ea men s. S alks we e cu in o single-eye se s and ea ed by imme sion o 48 h in cold
wa e (10°C) ollowed by 3h in ho wa e (50°C). Nex , se s we e plan ed in s e ile soil and
placed in mesh cage o p o ec hem agains insec s. Subsequen ly, hese plan s which ose
om hese cu ings we e es ed o he p esence o phy oplasma a e 2 and 6 mon hs and one
yea pos ge mina ion (Figu e.3.36).
Resul s
97
Figu e.3.36. Nes ed PCR esul s o he es plan s, which ecei ed cold and ho -wa e ea men a e 2
mon hs pos ge mina ion. Lane (1) con ains PCR p oduc ob ained om un ea ed suga cane (wi hou ho wa e
ea men ) (H65- 70 52). Lane (2) con ains PCR p oduc ob ained om un ea ed suga cane (H78- 77 50). Lane
(3) es plan (H65- 70 52) a e cold- and ho -wa e ea men (Aus alian ecipe). Lane (4) es plan (H78- 77
50) a e cold- and ho -wa e ea men (Aus alian ecipe). Re-ampli ica ion o aliquo o i s PCR wa e con ol
wi h nes ed p ime combina ion is in lane (5). DNA ladde is Fas Rule Middle Range (MBI Fe men as).
Figu e.3.37. Nes ed-PCR esul s o es plan s, which ecei ed cold and ho -wa e ea men a e 6
mon hs (a) and 1 yea (b) pos ge mina ion. Lane (1) ea ed plan (H65-70 52); lane (2) ea ed plan (H78-
77 50); lane (3) is posi i e con ol; e-ampli ica ion o aliquo s o i s PCR wa e con ol wi h nes ed p ime
combina ion a e in lanes (4-5); lane (M) GeneRule DNA ladde (MBI Fe men ans).
3.10.2. Ho wa e ea men wi h a ious du a ion
These es s we e ca ied ou in o de o in es iga e wha is he minimum imme sion ime a
50°C can elimina e he phy oplasma in in ec ed suga cane plan s. Fu he mo e, in hese es s
we didn’ soak he cu ings in cold wa e a 10°C be o e ho wa e ea men in o de o check
he in luence o lacking o cold wa e ea men on he ege a i e de elopmen . Thus, he
1
2
3
4
5
M
1
2
3
4
5
M
1
2
3
4
5
M
a
b
Resul s
104
Figu e.3.42. Compa ison be ween phloem sie e elemen s o whi e, a iega ed and g een lea es o whi e
lea phy oplasma-in ec ed suga cane. A: whi e lea pheno ype o suga cane whi e lea phy oplasma in ec ion,
whe e phy oplasmas a e clea ly isible in sie e elemen s (a ow). B: a iega ed lea pheno ype o suga cane
whi e lea phy oplasma in ec ion. Phy oplasmas a e ob ious p esen in sie e elemen s in inc eased numbe s in
compa ison wi h whi e lea (a ow). C: g een lea pheno ype o suga cane whi e lea phy oplasma in ec ion.
Phy oplasmas a e mo e abundan in some sie e elemen s han o he s (a ow). D: g een lea o unin ec ed
suga cane plan whe e i is used as con ol plan o compa ison. I is ob ious ha no phy oplasma is p esen in
sie e elemen s. In ec ed and unin ec ed lea es show he ypical sie e ana omy. Vacuoles and cy oplasm a e
used o a so called mic oplasm (Esau e al., 1965). Mos o he o ganelles a e absen , only ypical ound shaped
sie e elemen s plas ids a e p esen . Sie e elemen plas ids show he ypical pheno ype wi h c ys al inclusions
which a e common o poaeceae (a ow Fig. 53 D). Sie e ube (S), companion cell (CC), pa enchyma cell (PC).
Ba = 2 µm.
S
S
S
CC
CC
CC
CC
PC
S
CC
S
S
S
S

Resul s
105
Figu e.3.43. Compa ison be ween phloem companion cells o phy oplasma in ec ed and unin ec ed
suga cane. A: whi e lea pheno ype o phy oplasma-in ec ed suga cane whe e su ounding companion cell o
sie e elemen s con ains di e en o ganelles including acuoles and mi ochond ia bu hey don’ show any
phy oplasma. B: a iega ed lea pheno ype o phy oplasma-in ec ed suga cane show ypical companion cells
connec ed o each o he by plasmodesma a. Despi e o hese companion cells su ound sie e elemen which
con ain phy oplasmas bu hese cells a e phy oplasma- ee. C: g een lea pheno ype o phy oplasma in ec ion
whe e companion cell is also lack phy oplasma. D: g een lea o unin ec ed suga cane which used as con ol and
show pa ially one companion cell su ounds phy oplasma- ee sie e elemen .Mi ochond ia ha e a simila size
o phy oplasma bu could be clea ly dis inguished due o he p esence o C ys al o he inne mi ochond ial
memb ane. Sie e ube (S), Companion cell (CC), acuoles (V), plas id (P). Ba = 1 µm.
Resul s
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Figu e.3.44. Ul a hin sec ions o bundle shea h and mesophyll issues o phy oplasma-in ec ed suga cane.
A and B: a iega ed lea pheno ype o phy oplasma-in ec ed suga cane show ha phy oplasmas a e absen in he
bundle shea h issues. C and D: mesophyll issues o a iega ed lea pheno ype o phy oplasma-in ec ed
suga cane a e also phy oplasma- ee. Pa enchyma cells (PC), (A), (C) ba = 5 µm. (B), (D) ba = 2 µm.
3.13.3. Phy oplasma size and shape
Size o he phy oplasma bodies a ied om 200 nm o 800 nm (0.2 µm o 0.8 µm) in
diame e . Ou ansmission elec on mic oscopic s udies o whi e lea phy oplasma-in ec ed
suga cane lea showed sphe ical bodies which we e bounded by a poo ly de ined memb ane;
(Figu e.3.45).
Sie e ubes illed wi h nume ous phy oplasmas we e seen pa icula ly in
a iega ed lea es o diseased suga cane; (Figu e.3.46).
Resul s
107
Figu e.3.45. T ansmission elec on mic og aph o ypical memb ane-bound phy oplasma bodies which
p esen in sie e ube and con ain esembling DNA in sie e ube o whi e lea phy oplasma-in ec ed
suga cane lea . Sie e ube (S), phy oplasma (P). Ba = 0.2 µm.
Figu e.3.46. Ul a hin sec ions o phloem issue o phy oplasma-in ec ed suga cane. A: Ul a hin sec ion in
a iega ed lea pheno ype shows ha phy oplasmas (a ows) ill a phloem sie e elemen wi h a la ge numbe
which is app oxima ely mo e han 100 phy oplasma cells in one sie e ube. Phy oplasmas block he downwa d
ansloca ion pho osyn ha es and passing h ough a sie e-pla e po e lined wi h callose. B: highe magni ica ion
o he same las ul a hin sec ion. Sie e cell (S), companion cell (CC), pa enchyma cell (PC), sie e pla e (sp),
plas id (P), callose (CA). (A): ba = 2µm. (B): ba = 1µm.
S
P
P
P
CA
Resul s
108
3.13.4. Ul as uc u al changes o he phy oplasma in ec ion on lea e ana omy
Se e al ul as uc u al
changes we e obse ed on ul a hin sec ions o he ascula issues o
a ec ed suga cane plan s unde ansmission elec on mic oscope
(
TEM). Pa anchyma ic
cells o bundle shea h and mesophyll issue o a ec ed lea es showed some al e a ions
compa ing o unin ec ed lea es. In hese cells accumula ions o s a ch g anules and
plas oglobuli we e obse ed in whi e lea phy oplasma-in ec ed suga cane compa ing o
con ol unin ec ed one; (Figu e.3.47). Ou elec on mic oscopic s udies a e in ag eemen wi h
li e a u e whe e phy oplasma in ec ions led o a signi ican inc ease o s a ch in sou ce lea es
(Lepka e al. 1999). These da a a e consis en wi h ul as uc u al obse a ions epo ing
s a ch accumula ion in chlo oplas s associa ed wi h a se e e diso ganiza ion o hylakoids and
a educ ion in chlo ophyll con en (Muse i, 2006).
Figu e.3.47. Ul as uc u al compa ison be ween pa anchyma ic bundle shea h cells o in ec ed and
unin ec ed suga cane. A: a iega ed lea pheno ype o phy oplasma-in ec ed suga cane, whe e ul as uc u al
obse a ions indica e accumula ion o s a ch g anules (a ows) and plas oglobuli (head a ow) in chlo oplas s o
bundle shea h cells o in ec ed suga cane. B: g een lea o unin ec ed suga cane whe e i is used as con ol plan
o compa ison. I is clea ly ha accumula ion o s a ch g anules (a ows) in chlo oplas s o bundle shea h cells
is less han in ec ed suga cane. In addi ion, plas oglobuli a e no accumula ed in unin ec ed suga cane.
Pa enchyma cell (PC), plas id (P), nucleus (N), acuoles (V). Ba = 2µm.
Resul s
109
Figu e.3.48. Compa ison be ween chlo oplas s s uc u e o mesophyll cells in in ec ed and unin ec ed
suga cane. A: a iega ed lea o phy oplasma-in ec ed suga cane whe e an inc ease o plas oglobuli numbe and
size in diso ganized chlo oplas s was ound in mesophyll pa anchyma ic cells. B: g een lea o unin ec ed
suga cane which used as con ol, whe e mesophyll pa anchyma ic cells con ain no mal chlo oplas wi h lowe
o ma ion o plas oglobuli in compa ison wi h in ec ed suga cane. Plas id (P), plas oglobuli (a ows). Ba = 1µm.

Discussion
110
4. Discussion
4.1. Es ablishmen o he es o phy oplasma
Yellow lea synd ome (YLS) o suga cane has been associa ed wi h se e al bio ic and abio ic
causes du ing he pas ou decades. Lu e i uses and phy oplasmas a e wo ypes o plan
pa hogens ha a e ypi ied as causing symp oms o yellowing in hei hos s (Jones, 2002). I is
ha dly su p ising ha in suga cane he pa hogen canno be dis inguished by symp oms alone
(C onje e al., 1998; A ocha e al., 1999).
In he p esen s udy we ha e employed molecula -based ools o de ec ion and iden i ica ion
o he pu a i e causal agen o yellow lea synd ome and epo o he i s ime he p esence
o phy oplasma in Hawaiian suga cane cul i a s. The excep ional sensi i i y o PCR o e s
many ad an ages o de ec ion o plan pa hogens (He son and F ench, 1993). Applica ion o
his echnique o de ec ion and in es iga ion o phy oplasmas seems pa icula ly app op ia e
due o he small size o hese plan pa hogens and inabili y o cul u e hem in i o.
4.1.1. E iciency o PCR ampli ica ion
Phy oplasma diagnos ics and phylogene ics ha e his o ically been based on he 16S RNA
gene and he 16-23S RNA space egion because o he a ailabili y o uni e sal p ime s o
his egion (Hodge s and Dickinson, 2010). Nume ous PCR p ime combina ions ha e been
designed o diagnos ics and phylogene ics. Howe e , diagnos ics based on hese p ime s can
be p oblema ic, wi h occasional alse posi i es, pa icula ly h ough ampli ica ion o any
bacillus spp. ha migh be p esen in a plan sample (Ha ison e al., 2002). Howe e , based
on ou in es iga ion we ne e ound bacillus spp. in suga cane plan samples which was
con i med by RFLP analysis and DNA sequencing o nes ed-PCR p oduc s.
Though PCR is a ou ine echnique o phy oplasma de ec ion, he e s ill mee some
di icul ies, a leas wi h some p ime s which in some cases can induce dimme s, bands o non
speci ic sizes. In hese cases, alse posi i e esul s can be expec ed. Two ypes o con ol we e
he e o e ou inely applied in each PCR un o es possible gene a ion o alse posi i e
amplicons. One was he implemen a ion o wa e con ol o es he gene a ion o p ime
dimme s. Howe e , in ou hands, nes ed-PCR wi h all p ime combina ions used didn’
ampli y p oduc s om wa e used as empla e. The o he con ol expe imen was wi h
suga cane DNA om suga cane plan s which we e phy oplasma- ee, namely plan s om
Discussion
111
Egyp (G 549, Ph8013) and plan s which had unde gone ho wa e ea men . These
p epa a ions did no gi e a phy oplasma-speci ic amplicon which indica ed ha he suga cane
DNA does no con ain nucleo ide sequences which bind o he phy oplasma-speci ic p ime s.
In con as , in many cases he same phy oplasma-in ec ed suga cane samples ampli ied wi h
some p ime pai s ne e eac ed wi h o he p ime pai s, despi e he ac ha he used p ime s
we e uni e sal. In he case o no isible p oduc s we e ob ained om phy oplasma posi i e
samples, a highe dilu ion o DNA is used o dilu e he plan inhibi o s which may exis .
Howe e , hese phy oplasma posi i e samples ha e shown alse nega i e esul s a e dilu ion
oo. I seems ha in he case o phy oplasma posi i e samples, he p ime s p e e en ially
ampli ied phy oplasma sequence o expec ed size. Fo example, (P1/P7, R16F2n/R16R2) and
(R16mF2/R16mR1, R16F2n/R16R2) ha e been widely used o he de ec ion o phy oplasma
and a e p obably he mos ho oughly in es iga ed. They de ec all s ains o phy oplasmas
whe eas he DNA o non-in ec ed plan s does no eac . Many phy oplasma posi i e samples
we e alse nega i es wi h hese assays. The e o e, each suga cane sample was in es iga ed o
phy oplasma by di e en nes ed-PCR assays (I), (II), (III) and (IV) wi h di e en p ime pai
combina ions. Ou es s ha e showen signi ican di e ences in he esul s o he PCR assays
due o some weak o no ampli ica ion using pa icula p ime combina ions. Acco ding o ou
es s he p ime pai s used o nes ed-PCR assay III, (MLO-X/MLO-Y) and (P1/P2), which
ampli ied 16S-23S RNA space egion, was demons a ed o be he mos eliable one o
de ec he phy oplasma in suga cane plan s due o he high e iciency o PCR ampli ica ion,
high annealing empe a u e and low o no non-speci ic bands; in addi ion o he high
sensi i i y o hese p ime pai s whe e yielded s anda d p oduc s isualized in bands o a
s ong in ensi y. Ou analysis demons a ed di icul ies wi h he de ec ion abili y o
phy oplasma in suga cane plan s. In o de o explain he di e en esul pa e ns ob ained wi h
pa icula p ime combina ions, a subliminal amoun o empla e DNA, a p esence o PCR-
inhibi ing subs ances in DNA p epa a ions and sequen ial a iabili y o p ime a ge si es can
be aken in o accoun (Sk zeczkowski e al., 2001; Hein ich e al., 2001). Fo example, use o
he 16S-23S RNA space egion in ou in es iga ion was mo e eliable han 16S RNA gene
egion. I was mo e powe ul han he 16S RNA gene because i yielded s anda d p oduc s
isualized in bands o a s ong in ensi y as men ioned abo e.
As a consequence, in he case o c i ical samples, di e en p ime pai combina ions and also
sequencing should be used o elucida ion o phy oplasma p esence (F ano a, 2011).
Discussion
112
4.1.2. Ca y-o e con amina ion p oblems
The abili y o he PCR o ampli y minu e amoun s o empla e has he disad an age ha small
quan i ies o con amina ing DNA may be a p oblem o some applica ions like pa hogens
de ec ion. In gene al, he i e o phy oplasma in suga cane plan s is e y low and he s anda d
me hod is nes ed PCR, which enhances he sensi i i y o he es by wo successi e ounds o
ampli ica ion. I is a e y sensi i e me hod and he isk o alse posi i e signals is high. Two
ypes o wa e con ol we e he e o e ou inely applied o es o possible gene a ion o alse
posi i e amplicons. One was he implemen a ion o wa e con ol, whe e wa e was included
in he i s PCR- ound ins ead o DNA om suga cane lea es and hen he hypo he ical
amplicon was ans e ed o he second PCR- ound. In pa allel he second PCR- ound was
also pe o med wi h wa e ins ead o he i s - ound amplicon and se e al wa e con ols we e
used in each PCR ound. On he o he hand, i is impo an o ha e a designa ed clean a ea o
se ing up PCR eac ions om which o he DNA samples, especially PCR p oduc s, a e
excluded.
4.1.3. Q-PCR ( eal- ime PCR)
Pe o mance cha ac e is ics o used qPCR assay we e de e mined by ampli ying h ee
sepa a ely p epa ed se s o dilu ion se ies o h ee s anda d samples in wa e . All sys ems ga e
good alues as a as R², C , e iciency, limi o de ec ion and sensi i i y o amplicons which
showed a b oad dynamic ange ( i e log o de s o magni ude).
These pa ame e s we e also e alua ed o he a i icial samples ha imi a e in ec ed suga cane
samples. The calib a ion cu e o hese a i icial samples was e y impo an o check
sensi i i y o eal- ime PCR assay and o explain he alse nega i e esul s o qPCR o mos
ou suga cane samples. The i egula signals a he six h dilu ion o a i icial samples look
simila o he signals o expe imen al suga cane samples (Figu e 4.1). I is mos likely ha a
six h concen a ion le el (10¯⁵), using a 10- old dilu ion se ies o he phy oplasma-in ec ed
suga cane (phy oplasmal DNA) mixed wi h suga cane DNA o imi a e eal suga cane
samples, he i e o phy oplasmal DNA is e y low. I could be ha suga cane samples,
which con ain low i e o phy oplasmas canno be de ec ed sensi i ely by his di ec qPCR
assay due o he in luence o hos -ma e ial and ha may be ue o he suga cane plan s
which show yellow lea synd ome and con ain low i e o phy oplasma.
Discussion
113
Figu e.4.1. A: Log- iew o s anda d cu e cha . S anda d cu e de e mined a six concen a ion le els
( anging om 10⁰ o 10¯⁵) using 10- old dilu ion se ies o he phy oplasma-in ec ed suga cane (phy oplasmal
DNA) mixed wi h suga cane DNA o imi a e eal suga cane samples. Th eshold; is an a bi a y le el o
luo escence chosen on he basis o he baseline a iabili y. C ; is de ined as he ac ional PCR cycle numbe a
which he epo e luo escence is g ea e han he h eshold.∆Rn; is an inc emen o luo escen signal a each
ime poin . The ∆Rn alues a e plo ed e sus he cycle numbe . B: Log- iew o ampli ica ion cha o qPCR
esul s.
On he o he hand, i is essen ial ha he nucleic acid is su icien ly pu e o qPCR analysis.
Templa e con amina ion (i.e., p o ein, ca bohyd a es o o ganic sol en s) can ha e a huge
impac on assay eliabili y and ep oducibili y. We used high pu e PCR empla e p epa a ion
ki . Then he empla e DNA quali y was de e mined by Nanopho ome e . Since, diagnosis o
pa hogen in he plan s including suga cane is o en hampe ed by he p esence o PCR
inhibi o s such as polyphenolics, polysaccha ides and o he molecules ha may p oduce alse-
nega i e esul s e en some imes om hea ily in ec ed samples (Wein aub and Jones, 2010).
To p o e ha he absence o a signal is no due o such causes, p o ocols o con ol
ampli ica ion and de ec ion o he hos plan DNA ha e been de eloped such as 18S RNA
gene (Ch is ensen e al., 2004).
Acco ding o ou plan 18S RNA gene analysis, howe e , suga cane samples we e
su icien ly pu e o qPCR analysis. I appea s ha PCR inhibi o s can hampe diagnosis o
phy oplasma only when phy oplasmas exis in e y low i e as mos o ou suga cane
samples.
Ampli ica ion cha
Ampli ica ion cha
A
B
PCR cycle numbe
Th eshold
C
∆ Rn
PCR cycle numbe
∆ Rn
Plan DNA
phy oplasma DNA
Signals o six h dilu ion
Discussion
120
4.3. Iden i ica ion o he phy oplasma s ains in suga cane by phylogen ic analysis
Due o he inabili y o cul i a e phy olasmas in cell- ee media, molecula analyses o
conse ed gene sequences ha e become a ional means o phy oplasma axonomy and
classi ica ion. Use o DNA sequences o build up phylogene ic ees is widesp ead and
ecognized as a alid app oach o iden i ying axonomic ela ionships be ween o ganisms
(Hodge s and Dickinson, 2010).
Following decisions o phy oplasma axonomy aken by he Phy oplasma Wo king Team
du ing he 13 h In e na ional O ganiza ion o Mycoplasmology held in Fukuoka, Japan (14 o
19 July 2000) in gene al, a s ain can be desc ibed as a new “Candida us Phy oplasma
species” i i s 16S RNA gene sequence has less han 97.5% iden i y o any p e iously
desc ibed “Candida us Phy oplasma species.”
A BLAST sea ch o he 16S RNA gene sequences epo ed in his s udy showed ha hey
sha ed 99 o 100% sequence iden i y wi h hose o o he phy oplasmas in he as e yellows, X-
disease and ice yellow dwa g oups. This con i med ha he de ec ed phy oplasmas belong
o hese g oups o ‘Candida us phy oplasma’. Fo example, Egyp ian suga cane cul i a
G8447 con ains phy oplasma s ain (JN223446) clus e ed o he ice yellow dwa g oup,
closely oge he wi h so ghum g assy shoo phy oplasma om Aus alia (AF509324) wi h a
boo s ap alue o 81.1 and sha ed 99% sequence iden i y (Table.3.29 and Figu e.3.35.a).
Fu he mo e, i was p e iously epo ed ha he mo e dis an ly ela ed o SCGS agen , is he
so ghum g assy shoo (SGS) (Rao e al., 2007). The e o e, he Egyp ian suga cane cul i a
G8447 con ains phy oplasma s ain (JN223446) belongs o he ice yellow dwa g oup
‘Candida us phy oplasma o yzae’ and his s ain canno be desc ibed as a new “Candida us
Phy oplasma species” due o i s 16S RNA gene sequence has mo e han 97.5% iden i y o
any p e iously desc ibed “Candida us Phy oplasma species.”. Tha is also ue o
phy oplasma isola e (HM804282) om Cuban suga cane cul i a Ja605 clus e ed oge he
wi h o he s ains o X-disease g oup, among hem al eady epo ed suga cane yellows
phy oplasma s ain ound in Sou h A ica (AF056095) wi h a boo s ap alue o 48.7 and
sha ed 99% sequence iden i y (Table.3.29 and Figu e.3.35.a). The e o e, he Cuban suga cane
cul i a Ja605 con ains phy oplasma s ain (HM804282) belongs o he X-disease g oup
‘Candida us phy oplasma p uni’ and can’ be desc ibed as a new “Candida us Phy oplasma
species”.
O he Cuban suga cane cul i a C10-5173 was in ec ed wi h phy oplasma s ain (HQ116553)
clus e ed o he as e yellows g oup, closely oge he wi h suga cane yellows phy oplasma

Discussion
121
om B azil (EU423900) and maize bushy s un phy oplasma om Colombia (HQ530152)
wi h a boo s ap alue o 49.6 and sha ed 99% sequence iden i y (Table.3.29 and
Figu e.3.35.a). The e o e, he Cuban suga cane cul i a C10-5173 con ains phy oplasma
s ain (HQ116553) belongs o he As e yellows g oup ‘Candida us phy oplasma as e is’ and
his s ain can’ be also desc ibed as a new “Candida us Phy oplasma species”.
4.4. Ho wa e ea men in o de o ge phy oplasma ee plan
When a pa hogen is excluded om he p opaga ing ma e ial o a hos , i is o en possible o
g ow he hos ee o ha pa hogen o he es o i s li e (Aslam, 2001).
Ho wa e ea men has been p oposed o cu e do man woody plan ma e ial om
phy oplasmas. While issue cul u e echniques a e ou inely used o i us e adica ion; ew
epo s ha e been published on hei po en iali y in phy oplasma elimina ion (Dai e al., 1997;
Pa messu e al., 2002; Chalak e al., 2005). The e ec i eness o he me hod is based on he
ac ha do man plan o gans can wi hs and highe empe a u es han hose hei espec i e
pa hogens can su i e o a gi en ime (Ag ios, 2004).
The i s aim o ou ho wa e ea men was o ge nega i e con ol (phy oplasma- ee plan )
The e o e, we used an Aus alian ecipe (A ocha, 2005b), as long du a ion ea men (48 h in
cold wa e (10°C), ollowed by 3h in ho wa e (50°C)).The cu ings (app ox. 30 mm a e age
diame e ) we e kep in cold wa e be o e he ho wa e ea men was applied.
I seems ha , imme sion a 50°C o 30 min was no e ec i e o elimina e he phy oplasma
o ally om he cu ings bu i could be e ec i e in smalle diame e s. Fu he mo e, i could
be ha also depends on he i e o phy oplasmas in he plan ma e ial.
Ou es s showed ha he app op ia e ho wa e ea men , which ecommended o
phy oplasma elimina ion, is imme sion a 50°C o a leas 60 min. Fu he mo e, ou es s
showed ha he plan ma e ial (cu ings) should be he mally p epa ed o he ea men by
s o age o 48 hou s a 10°C in o de o p e en a poo ege a i e de elopmen especially o
long du a ion ea men a 50°C o 3 hou s. Due i could be ha his ea men could lead o
high mo ali y a es.
The ho wa e ea men which is p ac iced by he Hawaiian plan a ions o hei seed cane
ields (3 h a 50°C) is su icien o elimina e phy oplasma, whe eas he du a ion o he ho
wa e ea men o seed pieces which a e plan ed in he ields (20 min a 52°C) may be a he
ma gin o success ul bac e ia elimina ion.
Discussion
122
The e o e ield plan s we e es ed o phy oplasma wi h emphasis on he compa ison o g een
plan s wi h YLS-symp oma ic plan s, s anding side-by-side. Ou esul s showed ha hese
ield plan s we e in he case o a mos ly phy oplasma-in ec ed and i seems ha he e is no
clea associa ion be ween phy oplasma and symp oms due o some g een suga cane plan s
we e also posi i e o phy oplasma. Asymp oma ic suga cane was equen ly phy oplasma-
posi i e; his has also been epo ed by o he wo ke s (C onje e al., 1998a).
One explana ion o he poo co ela ion be ween phy oplasma and symp oms is ha some
phy oplasmas can exis in plan s wi hou e e causing disease o ha ing only a mino impac ,
as is he case o ash yellows in el e ash (Sinclai e al., 1994) and phy oplasmas in alde s
(Lede e and Seemulle , 1991), ap ico s (Ki kpa ick e al., 1990) and almonds (Uyemo o e
al., 1992). Such associa ions sugges ha he hos plan is ei he ole an o esis an o
phy oplasma in ec ion.
4.5. T ansmission es wi h suga cane aphid
Ou es s showed ha he suga cane aphids (Melanaphis saccha i) a e able o acqui e he
phy oplasmas because DNA ex ac ed om hese insec s p oduced an expec ed size o nes ed-
PCR p oduc . In addi ion, DNA sequencing o hese PCR p oduc s con i med ha . Ou es s
showed ha hese suga cane aphids a e able o acqui e he phy oplasmas bu hey a e unable
o ansmi he phy oplasmas in o he suga cane plan s because all a ge plan s (phy oplasma-
ee plan s) we e nega i e o phy oplasma in ec ion a e h ee mon hs pos inocula ion.
In ac , many aphids, whi e lies and mealy bugs a e phloem- eede s on plan species in ec ed
wi h phy oplasmas, bu so a none o hem has been ound o be a ec o o phy oplasmas.
Recen ly, apple aphids we e ound o be posi i e in PCR assays o apple p oli e a ion
phy oplasmas and we e suspec ed o be ec o s, bu he esul s o ansmission expe imen s
seem o exclude his possibili y (Cainelli e al., 2007). A phloem- eeding habi is hus
necessa y bu insu icien o phy oplasma ansmission.
I was an expec ed esul ha suga cane aphids a e unable o ansmi he phy oplasmas
(SCYP) because hus a , he e has been no epo o phy oplasma o spi oplasma
ansmission by a phloem- eeding aphid. The easons o lack o ansmission by aphids a e
no known. Si es o mollicu e a achmen o insec issues and o he pa hogen-insec
in e ac ions can be ci ed in a gene al sense o explain ansmission speci ici ies. Bu wha
molecula mechanisms, ha a e p esen in lea hoppe s and p esumably absen in aphids,
Discussion
123
accoun o he di e ences in mollicu e ansmission be ween hese majo insec g oups?
(Mish a, 2004). As a consequence, in e es ingly, aphids appa en ly do no se e as
phy oplasma ec o s.
4.6. T ansmission elec on mic oscopy o cy ological loca ion o phy oplasma
Phy oplasmas a e ans e ed wi h sali a o in ec ed insec ec o s in o he pie ced sie e
elemen , om which hey sp ead sys emically in he plan using he con inuous sie e ube
sys em due hey a e pleiomo ic and su icien ly small o pass eely h ough sie e po es.
Ou ansmission elec on mic oscopic s udies e ealed he p esence o suga cane whi e lea
phy oplasma only in phloem sie e ubes o diseased suga cane lea es bu no in cells adjacen
o he sie e elemen s including companion cells and phloem pa enchyma, al hough in many
cases he phy oplasmas ha e been eliably documen ed in companion cells and phloem
pa enchyma cells by elec on mic oscopy, as well as in sie e elemen s (Sille e al., 1987).
Se e al ul as uc u al changes we e obse ed unde ansmission elec on mic oscope
(TEM). Pa enchyma ic cells o bundle shea h and mesophyll issue o in ec ed lea es showed
some al e a ions compa ed o unin ec ed lea es. In hese cells accumula ions o s a ch
g anules and plas oglobuli we e obse ed in whi e lea phy oplasma-in ec ed suga cane
compa ed o unin ec ed con ol
(
Figu e.3.47). Ou elec on mic oscopic s udies a e in
ag eemen wi h li e a u e, whe e phy oplasma in ec ions led o a signi ican inc ease o s a ch
in sou ce lea es (Lepka e al., 1999). These da a a e consis en wi h ul as uc u al
obse a ions epo ing s a ch accumula ion in chlo oplas s associa ed wi h a se e e
diso ganiza ion o hylakoids and a educ ion in chlo ophyll con en (Muse i, 2006) due o
he dec ease o bo h Chl a and Chl b in lea es. “A dec ease in pho osyn he ic pigmen s has
been obse ed in maize plan s in ec ed wi h maize bushy s un (Junquei a e al., 2004), apples
in ec ed wi h apple p oli e a ion and g ape ine in ec ed wi h he bois noi phy oplasma. This
is p obably he esul o enhanced chlo ophyllase ac i i y in in ec ed lea es (Be amini e al.,
2002b) and i has been sugges ed ha phy oplasmas ha e a ole in he inhibi ion o
chlo ophyll biosyn hesis in plan hos lea es (Be amini e al., 2002a).”
“The descen o pho osyn hesis is he esul o phy oplasma in ec ion on pho osyn he ic
elec on anspo and enzyma ic ac i i ies, due o he loss o se e al hylakoid memb ane
p o eins and o he educ ion o lea soluble p o eins. These changes a e simila o hose
induced by lea ageing, so an in e e ence o phy oplasmas wi h plan ho mones ha egula e
senescence p ocesses in lea issues could be hypo hesized. In all kinds o diseases in which
Discussion
124
he e is des uc ion o lea issue like suga cane whi e lea phy oplasma, pho osyn hesis is
educed because he pho osyn he ic su ace o he plan is lessened. Mos i uses, mollicu e
diseases induce a ying deg ees o chlo osis and s un ing. In he majo i y o such diseases, he
pho osyn hesis o in ec ed plan s is educed g ea ly. In diseases caused by phy oplasmas,
bac e ia exis and ep oduce in he phloem sie e ubes, he eby in e e ing wi h he downwa d
ansloca ion o nu ien s” (Muse i, 2006).
An inc ease plas oglobuli numbe and size in diso ganized chlo oplas s was ound in
mesophyll pa anchyma ic cells o a iega ed lea o whi e lea phy oplasma-in ec ed
suga cane compa ed o g een lea o unin ec ed suga cane which was used as con ol
(
Figu e.3.48)
.
I is known ha cha ac e is ics o plas ids in senescen cells include educed
size, ounded shape and la ge plas oglobuli (Thomson and Pla -Aloia, 1987; Biswal and
Biswal, 1988; Noode´n, 1988).
As a consequence, he phy oplasma diseases a e complex and hei p og ess is also highly
a iable and depends on many ac o s including he s a e o he hos plan s, he pa hogen and
i s di e en bio ypes, he endency o mu a ion, he p esence and dynamics o he ec o s, he
i e o he phy oplasma, he en i onmen al condi ions as well as he ag onomical p ac ices
being used (Ciancio
and
Muke ji, 2008).
Summa y
125
5. Summa y
The Yellow lea synd ome (YLS) had been i s de ec ed and desc ibed in Hawaiian
suga cane plan a ions. The pole o i us Suga cane yellow lea i us was iden i ied as a causal
agen o he synd ome; howe e he e was no s ic co ela ion be ween he deg ee o
symp om exp ession and he i us i e. The e o e se e al su eys on b eeding s a ion
suga cane plan s in Hawaiian Islands we e done o Suga cane yellow lea phy oplasma
(SCYLP), a bac e ium which had been hypo hesized o be also a causal agen o YLS.
Two ypes o phy oplasmas we e ound in Hawaiian suga cane cul i a s mainly suga cane
whi e lea phy oplasma (SCWL) which is a membe in ice yellow dwa g oup, in addi ion o
as e yellows g oup. This was also ue o suga cane plan s om Hawaiian plan a ions, which
ou inely use ho wa e - ea men o he seed cane cu ings.
Suga cane samples we e ob ained also om o he coun ies including Cuba, Egyp , Sy ia and
Thailand whe e suga cane plan s a e also showing symp oms o yellowing o whi ing. As e
yellows and X-disease phy oplasmas we e ound in Cuban cul i a s whe eas one suga cane
cul i a om Egyp con ains g assy shoo phy oplasma ha is a membe in ice yellow dwa
g oup, bu he o he wo Egyp ian ones we e phy oplasma- ee. Sy ian suga cane was in ec ed
by phy oplasma ha iden i ied p elimina y in ice yellow dwa g oup. To ou knowledge, his
is he i s epo o he de ec ion and iden i ica ion o phy oplasma in suga cane plan s om
Hawaii, Egyp and Sy ia. Ou in es iga ion on Thai suga cane plan s was in ag eemen wi h
p e ious li e a u e whe e suga cane whi e lea (SCWL) phy oplasma is associa ed wi h whi e
lea disease (Nakashima e al., 1994; Wongkaew e al., 1997).
Q-PCR ( eal- ime PCR) o e s he oppo uni y o de ec he phy oplasma in a sensi i e,
speci ic and quick manne , bu ha is no ue o suga cane plan s wi h a e y low i e o
phy oplasma. The e o e, nes ed-PCR is be e han qPCR o low i e phy oplasma de ec ion
and ha is ue o suga cane yellow lea phy oplasma disease. A BLAST sea ch o he 16S
RNA gene sequences epo ed in his s udy showed ha hey sha ed 99 o 100% sequence
iden i y wi h hose o o he phy oplasmas in he As e yellows, X-disease and Rice yellow
dwa g oups. Howe e , no one o hese iden i ied s ains can be desc ibed as a new
“Candida us Phy oplasma species”. On he o he hand, Hawaiian suga cane cul i a H78-
7750
as a ep esen a i e
o
Hawaiian b eeding s a ion suga cane con ains
phy oplasma
clus e ed o s ain suga cane whi e lea (SCWL) phy oplasma, closely oge he wi h suga cane

Summa y
126
whi e lea phy oplasma om Taiwan (AY139874). I is possible o explain he occu ence o
(SCWL) phy oplasma in Hawaiian Islands, by insec ec o s o by in ec ed s em cu ings
which we e ob ained om o he coun ies. Thai suga cane con ains phy oplasma isola e
closely oge he wi h suga cane whi e lea phy oplasma om Myanma .
The ansmission elec on mic oscopic (TEM) s udies e ealed he p esence o suga cane
whi e lea phy oplasma only in phloem sie e ubes o diseased suga cane lea es bu no in
adjacen cells o he sie e elemen s including companion cells and phloem pa enchyma as
well. Acco ding o ul as uc u al
obse a ions unde TEM, pa enchyma ic cells o bundle
shea h and mesophyll issue o a ec ed lea es showed some al e a ions including
accumula ions o s a ch g anules, inc ease plas oglobuli numbe and size in diso ganized
chlo oplas s.
Insec ec o s o phy oplasmas a e phloem eede s. Thus a , none o aphid species has been
ound o be a ec o o phy oplasmas. Ou es s showed also ha black suga cane aphids
(Melanaphis Saccha i) we e unable o ansmi he phy oplasmas om in ec ed suga cane in o
he phy oplasma- ee one. Ho wa e ea men has been p oposed o cu e plan ma e ial om
phy oplasmas. Ou es s showed ha he app op ia e ho wa e ea men , which ecommended
o phy oplasma elimina ion, is imme sion o he suga cane s em cu ings a 50°C o 60 min.
Zusammen assung
127
6. Zusammen assung
Das Yellow Lea Synd om (YLS) bei Zucke oh wu de zue s in Plan agen Hawaiis en deck
und on do besch ieben. Das Pole o i us Suga cane Yellow Lea Vi us konn e als
e u sachendes Agens des Synd oms iden i izie we den, jedoch gab es keinen s ik en
Zusammenhang zwischen de In ensi ä de Symp ome und dem Vi us i e . Deshalb wu den
Analysen an Zucke oh p lanzen aus de hawaiianischen Zuch s a ion du chge üh , um die
P lanzen au Suga cane yellow lea phy oplasma (SCYLP) zu es en, einem Bak e ium, das
eben alls als mögliche Auslöse on YLS e mu e wu de.
Zwei Typen on Phy oplasma wu den in den hawaiianischen Zucke oh kul i a en en deck ,
nämlich Suga cane Whi e Lea Phy oplasma (SCWL), ein S amm de Rice Yellow Dwa
G uppe, und ein S amm de As e Yellows G uppe. Dies gal auch ü Zucke oh p lanzen aus
hawaiianischen Plan agen, obwohl bei diesen ou inemäßig eine Heißwasse -Behandlung
ih e Se zlinge, welche Phy oplasma eliminie en könn e, du chge üh wi d.
P oben on Zucke oh p lanzen ande e Lände (Kuba, Ägyp en, Sy ien und Thailand), in
denen P lanzen mi Ve gilbungs- ode Bleichungssymp omen es ges ell we den, konn en
eben alls ge es e . As e Yellows und X-Disease Phy oplasmen and man in kubanischen
Kul i a en, wäh end ein ägyp isches Kul i a G assy Shoo Phy oplasma (eben alls ein
S amm de Rice Yellow Dwa G uppe) en hiel . Zwei ande e Kul i a e aus Ägyp en wa en
phy oplasma ei. Auch das sy ische Zucke oh wa on einem Phy oplasma de Rice Yellow
Dwa G uppe in izie . Unse es Wissens sind das die e s en Nachweise on Phy oplasma in
Zucke oh aus Hawaii, Ägyp en und Sy ien. Die Analysen an hailändischen P lanzen
bes ä ig en publizie e E gebnisse, dass mi Suga cane Whi e Lea (SCWL) Phy oplasma
in izie e P lanzen mi Whi e Lea Disease in Zusammenhang s ehen (Nakashima e al., 1994;
Wongkaew e al., 1997).
Q-PCR ( eal- ime PCR) gil als emp indliche, spezi ische und asche Me hode um
Phy oplasma in P lanzenma e ial zu messen, dies e wies sich abe o ensich lich nich ü
Zucke oh mi nied igem Phy oplasma-Ti e . Deshalb wu de nes ed-PCR als die sensi i e e
Me hode, um Phy oplasma-In ek ion nied igen Ti e s bei Zucke oh es zus ellen,
angewand . Ein BLAST-sea ch zeig e, dass die 16S RNA de ge undenen Phy oplasma-
S ämme 99-100% Sequenziden i ä mi Phy oplasmen de As e Yellows, X-Disease und Rice
Yellow Dwa G uppen au weisen, sodass keine da on als neue “Candida us phy oplasma
Zusammen assung
128
A ” besch ieben we den kann. Das Phy oplasma aus dem komme ziellen hawaiianischen
Kul i a H78-7750 g uppie e sich in Suga cane Whi e Lea Phy oplasma (SCWLP) ein,
zusammen mi einem S amm aus Taiwan. Es e schein also möglich, dass übe Insek en als
Vek o en ode in izie e Se zlinge Phy oplasma aus Taiwan nach Hawaii kam ode
umgekeh . Das hailändische Phy oplasma s eh am nächs en dem Whi e Lea Phy oplasma
aus Myanma .
Gewebeschni e im T ansmissions-Elek onenmik oskop (TEM) zeig en, dass Phy oplasma
ausschließlich in den Sieb öh en de Lei bündel zu inden is , nich in Gelei zellen,
Phloempa enchym ode ande en Bla zellen. Die no male weise g ünen Gewebe de
in izie en Blä e (Bündelscheide und Mesophyll) zeig en s a ke zy ologische Ve ände ungen
wie Akkumula ion on S ä kekö ne n, eine g oße Anzahl on Plas oglobuli und
deso ganisie e S uk u en in Chlo oplas en.
Vek o en ü Phy oplasma sind Phloemsauge , jedoch wu de bishe keine Bla laus als Vek o
nachgewiesen. Es konn e gezeig we den, dass die schwa ze Zucke oh laus Melanaphis
saccha i, die de wich igs e Vek o ü Suga cane Yellow Lea Vi us is , Phy oplasma nich
übe agen kann. Heißwasse -Behandlung wa als Me hode zum Ab ö en on Phy oplasma in
P lanzen eilen besch ieben wo den. Dies konn e bes ä ig we den und eine 60-minü ige
Behandlung in 50° heißem Wasse kann ü die Eliminie ung on Phy oplasma in
Zucke oh se zlingen emp ohlen we den.
Acknowledgemen
129
7. Acknowledgemen
Fi s o all, I would like o acknowledge my supe iso P o . D . Ewald Komo , who ga e me
he accep ance o come o Uni e si y o Bay eu h and o wo k in his lab.
Thanks o all people in my depa men (plan physiology) o hei help, especially P o . D .
S ephan Clemens, Ch is iane Meinen and U sula Fe e a.
I would like o hank D . E ic Hummel, Daniel Souza, Ch is ian Seybold, Philipp Gasch and
Thomas Liebens ein; hey a e no only colleagues bu nice iends who kindly helped me wi h
all kinds o si ua ions.
I app ecia e he assis ance om
D . Al ons Weig o do phylogene ic analysis.
I am g a e ul o Ri a G o jahn ( echnician in he labo a o y o elec on mic oscopy) o
p epa a ion o all ul a hin sec ions.
This is he igh place o hank P o . D . A. Be accini a Uni e si y o Bologna, I aly; P o .
D . E. Seemülle a ins i u e o plan p o ec ion in Dossenheim, Ge many and D . J. Hodge s
a Uni e si y o No ingham, UK o hei coope a ion and ad ices h oughou my wo king
p ocess.
My schola ship was unded by Uni e si y o Aleppo, Sy ia. I am e y hank ul o his
suppo .
I would like o hank my supe iso D . Nada Omlah a Uni e si y o Aleppo, Sy ia.
I am ex emely hank ul o my pa en s o hei lo e and ca e which gi es me he g ea es
mo i a ion in li e and o hei encou agemen wi h e e y s ep I ake.
And God,
I am o e e e y hank ul o being wi h me all he ime and help me wi h you bes
blessings.
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Lis o igu es
145
9. Lis o igu es
1. In oduc ion
1
Figu e.1.1. Compa ison o sizes o some eubac e ia. 3
Figu e.1.2. Pleomo phic phy oplasmas in sie e ubes. 4
Figu e.1.3. Phy oplasmas and hei diseases a e wo ldwide. 6
Figu e.1.4. Hos cycle o phy oplasmas. 7
Figu e.1.5. Phy oplasmas a e i micu es. 8
Figu e.1.6. Diag am o he longi udinal iew o phloem cells. 11
Figu e.1.7. Suga cane yellow lea synd ome (YLS). 12
Figu e.1.8. Suga cane whi e lea (SCWL). 13
2. Ma e ial and Me hods
17
Figu e.2.1. A diag am illus a ing o he me hod o nes ed PCR. 25
Figu e.2.2. Diag amma ic ep esen a ion o loca ion o used p ime pai s and expec ed size o hei
ampli ied p oduc s based on phy oplasma RNA ope on. 29
Figu e.2.3. Diag amma ic ep esen a ion o a phy oplasma RNA ope on and genomic loca ion o p ime s
used o phy oplasma de ec ion. 29
Figu e.2.4. Diag am illus a ing o SYBR G een du ing PCR ampli ica ion. 35
Figu e.2.5. Diag am illus a ing o TaqMan p obe chemis y mechanism. 36
Figu e.2.6. Diag amma ic ep esen a ion o genomic loca ion o qPCR p ime s and p obe used o
phy oplasma de ec ion. 37
3. Resul s
39
Figu e.3.1. Nes ed PCR-p oduc s o posi i e and nega i e con ols and o a posi i e con ol, which was
mixed wi h inc easing amoun s o suga cane DNA. 40
Figu e.3.2. Phy oplasma in Hawaiian and Cuban suga cane cul i a s. 43
Figu e.3.3. Phy oplasma in Egyp ian and Sy ian suga cane cul i a s. 43
Figu e.3.4. Res ic ion agmen analysis o PCR p oduc s om Hawaiian and Cuban suga cane cul i a s
con aining phy oplasma. 45
Figu e.3.5. Nes ed-PCR assay (II) p oduc s (1.2kb) ampli ied wi h p ime s (SN910601/P6,
R16F2n/R16R2). 47
Figu e.3.6. RFLP p o iles o nes ed-PCR assay (II) p oduc s. 48
Figu e.3.7. Nes ed-PCR assay (III) p oduc s (0.2kb) ampli ied wi h p ime pai (MLO-X/MLO-Y, P1/P2).
49
Figu e.3.8. Nes ed-PCR assay (IV) p oduc s. 50