Nedavno opisana svojta Candidatus phytoplasma solani (CPs), poznatija pod imenom stolbur fitoplazma, jedna je od fitoplazma s najširim krugom prirodnih domaćinskih vrsta koji uključuje različite ...zeljaste i drvenaste biljke. Brojne korovne biljke imaju ulogu alternativnih domaćina, a ujedno su značajan rezervoar CPs‐a za infekciju kukaca vektora kojima se ova fitoplazma i prenosi. CPs je uzročnik mnogih ekonomski značajnih bolesti bilja, među kojima su najistaknutije žutica vinove loze crno drvo (bois noir), crvenilo kukuruza (maize redness), propadanje lavande (yellow decline of lavender) te stolbur krumpira. U članku su ukratko opisane spomenute bolesti s naglaskom na simptomatologiju i epidemiologiju te mjere zaštite.
Uglata pjegavost lista jagode koju uzrokuje bakterija Xanthomonas fragariae može izazvati znatne gubitke u proizvodnji jagode. Nakon što je prvi put opisana u SAD-u 1960-ih, u idućih 15 godina ...pojavila se na svim kontinentima u zemljama gdje se uzgaja jagoda. Uz tu bolest, jagode mogu biti zaražene i fitoplazmama iz više od deset ribosomskih skupina i podskupina. Iako fitoplazmoze i bakterioze najčešće nisu razorne bolesti, uzrokuju znatne gubitke ponajprije zbog lošeg izgleda i kakvoće ploda koji nije prihvatljiv za tržište.
The plant samples with symptoms typical for phytoplasma infection were
collected in Serbia from 2009 till 2011. In those samples phytoplasmas were
detected by PCR technique with universal primers ...P1/P7 and R16F2n/R16R2 and
identified according to their Tru1I, HhaI, RsaI and Tsp509I restriction
profiles of digested R16F2n/R16R2 amplicons as Aster yellows phytoplasma
(ribosomal group 16SrI), subgroups 16SrI-B, I-C and I-P or as Stolbur
phytoplasma (ribosomal group 16SrXII). Applying the same PCR-RFLP method on
19 strains of Aster yellows phytoplasmas from collection and three strains
from carrot from Serbia, it was confirmed that this strains belong to
subgroups 16SrI-A, I-B, I-C and I-F as stated in literature. Also the
subgroups were determined for four strains from collection that are used in
analyses for the first time and that have no literature data. Using newly
designed primers AYgroesF/AYampR and AYgroelF/AYgroelR, the groEL gene was
successfully amplified in all 34 strains of Aster yellows phytoplasma tested.
RFLP analyses of AYgroelF/AYgroelR amplicons with Tru1I i AluI restriction
enzymes revealed existence of six and eight different restriction profiles,
respectively, according to which all tested strains were classified in nine
groELI subgroups (groELI-I till groELI-IX). On the basis of groEL gene,
subgroups 16SrI-A and I-C were further differentiated into two groELI
subgroups, 16SrI-B into three, while subgroups 16SrI-M and I-L showed no
difference to some strains belonging to subgroup 16SrI-B. Subgroups 16SrI-F
and I-P could be differentiated from other subgroups on the basis of groEL
gene as on the basis of 16S rDNA. The seven newly detected Aster yellows
strains from Serbia were affiliated to subgroups groELI-III, I-VII and I-IX.
RFLP analyses with MboI, Tsp509I and Tru1I restriction enzymes of tuf gene,
HhaI, AluI and Tsp509I of rp gene and Tsp509I and Tru1I of secY gene,
classified 22 selected Aster yellows strains into six tufI subgroups and
seven rpI and secYI subgroups. Subgroup 16SrI-A was further differentiated
into two tufI subgroups and subgroups 16SrI-A and I-B were further
differentiated into two rpI and secYI subgroups each. On the other hand
subgroups 16SrI-B, I-M and I-L showed no mutual differences, while subgroups
16SrI-C, I-F and I-P could be differentiated from other subgroups on the
basis of all three genes tested as on the basis of 16S rDNA. RFLP analysis of
tuf gen with HpaII restriction enzyme showed that all 116 tested Stolbur
phytoplasma strains from Serbia belong to tuf type II variant. For further
analyses, 39 Stolbur strains were selected out of 116 detected and two
strains from grapevine from Croatia were also included in analyses as tuf
type I reference strains to gain a larger picture of variability of groEL
gene in Stolbur phytoplasmas. In all 41 Stolbur tested strains, groEL gene
was successfully amplified with newly designed primers STOLgroesF/STOLstampR
and AYgroelF/STOLgroelR2. RFLP analysis of these amplicons with Tru1I
restriction enzyme determined the presence of two different profiles.
Analysis of groEL gene sequences of eight selected strains, including tuf
type I strains, revealed the presence of three SNPs leading to determination
of five genotypes in total. First SNP is inside groES gen and it is a
synonymous one resulting in no predicted amino acid difference, while two
others are inside the groEL gen and they lead to different predicted amino
acid sequences. Also it can be noticed that in the predicted amino acid
sequences at the position 338 all strains from Serbia, belonging to tuf type
II variant, have alanine (A), while two strains from Croatia, tuf type I
variants, have threonine (T). Applying AYgroelF, AYgroelR and STOLstampR
primers together in multiplex PCR reaction, it was possible to detect only
Aster yellows and Stolbur phytoplasma, or both together, in positive samples
from collection as well as in field collected samples. Analyzing groEL gene
sequences of 27 Aster yellows tested phytoplasma strains, it has been
established that whole groEL gene was 1611 bp long (protein consisted of 536
amino acids), that there were 144 polymorphic positions in total, 55 of them
resulted in different amino acid when translated while 89 was synonymous
ones, that among the observed strains there was difference in 50 amino acids
in protein sequences, that subgroup 16SrI-A is the most variable one, that
equatorial domain of GroEL consisted of residues 5-132 and 406-521, apical
domain of 189-373 and intermediate domain consisted of residues 133-188 and
374-405. Also, it has been determined that in GroEL protein of AY
phytoplasmas there were some differences in amino acid composition in
positions that are, from literature, important for its role in folding other
proteins. Comparative sequence analyses of 16S rRNA and groEL gene revealed
that the average nucleic acid similarity of examined AY strains was 99.5% and
98.1%, respectively, ranging from 98.7 till 100% for 16S rRNA and from 93.7
till 100% for groEL gen. Average amino acid similarity of GroEL protein of AY
phytoplasmas tested was 97.8% ranging from 94.6 till 100%. Analysis of
sequences of tuf, rp and secY genes determined that average nucleic acid
similarity of examined AY strains for tuf gene was 97.2% (93-99.9%), for rp
97.4% (93.2-100%) and for secY gene it was 94.8% (84-100%). The lowest
nucleic acid similarity regarding all five observed genes had strain PopD
(ribosomal subgroup 16SrI-P) in respect to some of the strains from subgroups
16SrI-A or I-B. Phylogenetic tree reconstructed on the basis of nucleic acid
sequences of whole groEL gene had basically the similar topology like the one
made on the basis of 16S rDNA sequences, with the difference that new
clusters inside subgroups 16SrI-A, I-B and I-C have emerged. Strain AY-A,
which belongs to subgroup 16SrI-F, was situated as a separate lineage, while
the strain PopD (16SrI-P) was separated forming the new lineage outside all
the other strains which clustered together. Nine lineages delineated on this
phylogenetic tree were in agreement with the affiliation to groELI subgroups,
made on the basis of RFLP analyses. Phylogenetic tree reconstructed on the
basis of tuf, rp and secY gene sequences put together had identical topology
as the one made on the basis of groEL gene sequences. On the basis of
PCR-RFLP method it has been shown that groEL gene is more informative in
regard to 16S rRNA, tuf, rp and secY genes and by phylogenetic analyses it
has been confirmed that groEL gene has lower intergroup sequence similarities
than 16S rDNA and therefore has more resolution power in separating closely
related strains. These results show that usage of groEL gene and its analyses
with PCR-RFLP method is reliable tool for detection as well as for
identification of Aster yellows phytoplasma strains from collection and from
field collected samples.
Na teritoriji Republike Srbije su u periodu od 2009. do 2011. godine
sakupljeni uzorci biljaka sa simptomima karakterističnim za prisustvo
fitoplazmi. Upotrebom PCR metode pomoću univerzalnih prajmera P1/P7 i
R16F2n/R16R2 u njima su detektovane fitoplazme. Upotrebom restrikcionih
enzima Tru1I, HhaI, RsaI i Tsp509I na R16F2n/R16R2 amplikone, ove fitoplazme
su na osnovu dobijenih restrikcionih profila identifikovane kao Aster yellows
fitoplazme (ribozomalna grupa 16SrI), podgrupe 16SrI-B, I-C i I-P, odnosno
kao Stolbur fitoplazme (ribozomalna grupa 16SrXII). Upotrebom iste PCR-RFLP
metode kod 19 sojeva Aster yellows fitoplazmi iz kolekcije i tri soja
poreklom iz šargarepe iz Srbije, potvrđena je pripadnost podgrupama 16SrI-A,
I-B, I-C i I-F preuzeta iz literature. Takođe je utvrđena pripadnost
određenoj podgrupi kod četiri soja iz kolekcije, koji se prvi put koriste u
analizi i o kojima nema literaturnih podataka. Upotrebom novodizajniranih
prajmera AYgroesF/AYampR i AYgroelF/AYgroelR umnožen je groEL gen kod sva 34
testirana soja Aster yellows fitoplazmi. Analizom restrikcionih profila
dobijenih pomoću Tru1I i AluI restrikcionih enzima na AYgroelF/R amplikone,
utvrđeno je postojanje šest, odnosno osam različitih profila na osnovu kojih
su svi testirani sojevi svrstani u devet groELI podgrupa (groELI-I do
groELI-IX). Na osnovu groEL gena podgrupe 16SrI-A i I-C su dalje
diferencirane u dve groELI podgrupe, 16SrI-B u tri, dok podgrupe 16SrI-M i
I-L nisu pokazale nikakve međusobne razlike u odnosu na jedan deo podgrupe
16SrI-B. Podgrupe 16SrI-F i I-P se razlikuju od ostalih podgrupa i na nivou
groEL gena kao i na nivou 16S rDNK. Takođe je utvrđeno da sedam
novodetektovanih sojeva Aster yellows fitoplazmi iz Srbije pripada podgrupama
groELI-III, I-VII i I-IX. Analizom tuf gena, dela rp operona i secY gena kod
22 odabrana od 34 testirana soja soja Aster yellows fitoplazmi moguće je
testirane sojeve klasifikovati u šest tufI i sedam rpI i secYI podgrupa na
osnovu MboI, Tsp509I i Tru1I restrikcionih profila za tuf gen, HhaI, AluI i
Tsp509I za rp gen i Tsp509I i Tru1I za secY gen. Podgrupa 16SrI-A je dalje
diferencirana u dve tufI podgrupe, odnosno 16SrI-A i I-B u po dve rpI i secYI
podgrupe, dok podgrupe 16SrI-B, I-M i I-L nisu pokazale nikakve međusobne
razlike. Podgrupe 16SrI-C, I-F i I-P se razlikuju od ostalih podgrupa i na
nivou sva tri testirana gena kao i na nivou 16S rDNK. Analizom tuf gena kod
116 detektovanih sojeva Stolbur fitoplazmi iz Srbije, pomoću HpaII
restrikcionog enzima, utvrĎeno je da svi testirani sojevi pripadaju tuf type
II podgrupi. Od ovih uzoraka je za dalju analizu groEL gena odabrano 39
sojeva, a radi kompletnije slike o varijabilnosti groEL gena kod Stolbur
fitoplazmi, u analizu su uključena i dva soja Stolbur fitoplazmi iz Hrvatske,
poreklom sa vinove loze, kao kontrolni sojevi Stolbur fitoplazmi tuf type I
podgrupe. Kod sva 41 testirana uzorka Stolbur fitoplazmi uspešno je umnožen
groEL gen upotrebom novodizajniranih prajmera STOLgroesF/STOLstampR i
AYgroelF/STOLgroelR2. Analizom dobijenih fra
SAŽETAK
U uzgoju loze fauna cvrčaka predstavlja značajnu skupinu insekata. Od vektora fitoplazmi loze značajni su insekti koji se hrane iz floemskog tkiva biljaka kao što su cikade iz porodica: ...Cicadelidae, Coccidae, Fulgoridae i Psyilloidaea, a fitoplazme prenose na perzistentan način. Tijekom 2005. i 2006. godine u vinogradima Istre obavljena su istraživanja faune cvrčaka, florističkog sastava korova i biljaka domaćina fitoplazmi vinovoe loze. Istraživanja su obavljana na 11 lokacija u 10 vinograda. Sakupljanje cvrčaka obavljeno je entomološkom mrežom, pomoću žutih ljepljivih ploća, metodom udaraca i ekshaustorom. Sakupljeni cvrčci determinirani su pomoću binokulara i ključeva za determinaciju vrsta, dok je nazočnost fitoplazmi BN i FD utvrđivana pomoću molekularnih analiza (PCR, RFLP). Popis i determinacija florističkog sastav korova obavljao se neposredno u istraživanim vinogradima pomoću ključeva za determinaciju vrsta. Uzorci biljnog materijala za analizu PCR metodom uzimani su na temelju vizualnog pregleda i tipičnih simptoma koje izazivaju fitoplazme. Determinacija cvrčaka i molekularne analize obavljane su u Italiji u Istituto Sperimentale per la Viticultura – Conegliano. Tijekom istraživanja sakupljena su 243 uzorka insekata. Od navedenog broja u 207 uzoraka utvrđene su cikade iz 40 rodova. Popisom florističkog sastava korova utvrđeno je 105 vrsta iz 36 porodica. U navedeni popis uvrštene su i vrste Corylus avellana L i Clematis vitalba L, vrste koje su pokazivale znakove oboljenja fitoplazmi i nalazile su se neposredno uz istraživane vinograde. Nad istima je obavljena analiza na prisutnost fitoplazmi FD i BN. Molekularna istraživanja PCR metodom na prisustvo fitoplazmi FD i BN obavljena su nad 34 uzorka insekata i 22 biljna uzorka. Od navedenih uzoraka niti jedan uzorak insekata nije bio pozitivan na FD i BN. U lišću vinove loze sorte malvazija iz vinograda u Novigradu i Starićima utvrđena je fitoplazma BN. U uzorku lišća C. vitalba na lokaciji Barat utvrđena je fitoplazma FD – tip C. Rezultati provedenih istraživanja ukazuju da su u vinogradima Istre prisutne vrste cvrčaka koje su poznati vektori fitoplazmi vinove loze. Njihovo prisutsvo u vinogradima Istre predstavljaju potencijalnu opasnost u širenju fitoplazmi s obzirom da je BN pronađen na lozi, a FD na korovskoj vrsti C. vitalba.
Fitoplazmoze vinove loze Kozina, Bernard; Karoglan, Marko; Mihaljević, Marin
Glasnik Zaštite Bilja,
12/2008, Letnik:
31, Številka:
6
Paper
Odprti dostop
Fitoplazme, ranije nazivane ‘’mikoplazmama slični organizmi’’, uzročnici su nekoliko stotina biljnih bolesti uključujući i žutice vinove loze. Na raznim kultivarima vinove loze u vinogradima diljem ...Hrvatske potvrđene su fitoplazme iz skupine stolbur (16SrXII-A). Simptomi fitoplazmoza uočeni su i na području zagrebačkog vinogorja, naročito na kultivaru Chardonnay. Opisani su karakteristični simptomi zaraze fitoplazmama, osjetljivost pojedinih kultivara vinove loze, prijenos zaraze i najznačajniji vektori te geografska rasprostranjenost i način suzbijanja fitoplazmoza.
Masline mogu inficirati četiri različite vrste fitoplazmi koje mogu imati značajan utjecaj na njihov rast i razvoj to prinose. Pored prijenosa putem kukaca dominantan način njihovog širenja ...predstavlja korištenje zaraženog sadnog materijala. Članak daje kratak pregled simptoma koje fitoplazme mogu uzrokovati na zaraženim stablima te mogućnosti njihove detekcije i kontrole.
