Download Diagnostic Methods for Bacterial Blight of Grape Xylophilus

Diagnostic Methods
for
Bacterial Blight of Grape
Xylophilus ampelinus
This Diagnostic Protocol can be constantly updated and is only correct at time of printing
(Thursday 4th of May 2017 at 09:51:55 AM).
The website http://www.padil.gov.au/pbt should be consulted to ensure you have the most
current version before relying on the information contained.
Introduction
Bacterial blight of grapevine is a serious, chronic and destructive vascular disease of grapevine,
affecting commercially important cultivars. It is widespread in the Mediterranean region and
South Africa and may occur in other regions. The pathogen survives in the vascular tissues of
infected plants.
Biology
Stages of Development
Xylophilus ampelinus is a strictly aerobic, non-spore forming, Gram-positive rod, 0.5-0.8 x 1.63.2µ, ave. 0.6 x 2.3µ, motile by one polar flagellum. Occasionally isolates have some
filamentous cells 8-10 times longer than usual. Occurs singly or in pairs.
It produces a yellow insoluble pigment and metabolizes sugars oxidatively.
Primary infections occur mainly on shoots 1 or 2 years old, via leaves, blossoms and grapes.
Host Range
The only known host of Bacterial blight is Vitis vinifera (Grapevine).
Distribution
Bacterial blight is known to occur in Asia, South Africa, Greece, France, Spain, Italy, Turkey,
Portugal, USSR, South America & the Canary Islands.
Distribution map - June 2006 - see attached PDF file.
Distribution map: October 1999 (Edition 3), Map # 531
http://217.154.120.6/PLANTDISEASES/showarticle.nsp?lastquery=xylophilus+ampelinus&sortfield=NONE&docindex=0&ranked=1&var_it
emsfound=2&var_itemsallowed=2&var_backindex=1
Distribution map: March 1986 (Edition 2), Map # 531 -
http://217.154.120.06/CABDirect/showarticle.nsp?lastquery=xanthomonas+ampelina&sortfield=NONE&docindex=1&ranked=2&var
_itemsfound=37&var_itemsallowed=37&var_backindex=1
Transmission
Most information is based on observational data rather than experiments.
Epidemiology of bacterial blight indicates that no insect vector of importance has been found.
The major sources of infection are apparently infected propagating material and epiphytic
bacteria that enter through wounds.
Bacteria overwinter in the vines, emerge, probably in spring and are carried to healthy shoots,
most probably in wind and rain. Wounds may facilitate entry but are not needed for primary
infection.
Considerable spread can occur via propagating material, grafting & pruning. Bleeding sap
appears to be an important source of contamination. Illegally imported plants pose the greatest
risk & if such material is infected, the disease is likely to become established.
The disease is associated with warm moist conditions, and spread is favoured by overhead
sprinkler irrigation. From initial disease foci, local spread in vineyards tends to occur along
rows.
Dispersal
Spread can occur via propagating material, grafting & pruning. Bleeding sap appears to be an
important source of contamination. Illegally imported plants pose the greatest risk & if such
material is infected, the disease is likely to become established.
The disease is associated with warm moist conditions, and spread is favoured by overhead
sprinkler irrigation. From initial disease foci, local spread in vineyards tends to occur along
rows.
- Survival
The bacterium overwinters in the vines, emerges in spring & is carried to healthy shoots mainly
by rain splash. Shoots are susceptible to infection during autumn and winter and non-susceptible
during spring & summer. The bacterium is able to survive in wood, and thus may be transmitted
between locations in infected cuttings.
The ability of X. ampelinus to survive for several years inside plants without inducing symptoms
may result in a latency period.
Effect
Losses arise from reduced productivity & shortened life of diseased vines. Some cultivars are
more susceptible than others & no control measures are known.
Bacterial blight is a chronic, systemic disease of significant economic importance.
Taxonomy
Classification
Phylum Proteobacteria
Order Xanthomonadales
Family Xanthomonadaceae
Genus Xylophilus
Species ampelinus
Name and Synonyms
Synonyms:
Xanthomonas ampelina Panagopoulos
Xylophilus ampelinus (Panagopoulos)
Common names:
Bacterial blight of grapevine
Tsilik marasi in Greece
Maladie d'Oleron, Nécrose bactérienne de la vigne in France
Vlamsiekte in South Africa
Mal nero in Italy
Identification Keys
Xanthomonas ampelina is an anomalous member of the genus Xanthomonas which is readily
distinguished on the basis of a few tests. The pathogen may be rapidly detected in grapevine sap
using the indirect immunofluorescence technique.
Detection
Symptom Description
Bacterial necrosis of grapevines is characterized by typical symptoms such as cankers on stems
and petioles, by necrotic foliar spots and by bud death.
In early spring, buds on infected spurs fail to open, or make stunted growth which eventually
dies. Affected spurs often appear slightly swollen because of hyperplasia of the cambial tissue.
Cracks appear along such spurs, become deeper and longer, forming cankers. Young shoots may
develop pale yellowish-green spots on the lowest internodes. These expand upwards on the
shoot, darken, crack and develop into cankers. Cracks, and later cankers, also form on more
woody branches later in spring. In summer, cankers are often seen on the sides of petioles,
causing a characteristic one-sided necrosis of the leaf. They may also appear on main and
secondary flower and fruit stalks. Leaf spots and marginal necrosis occur sometimes. Gum
formation is not necessarily a symptom.
Infection usually occurs on the lower two to three nodes of shoots that are 12 - 30 cm long, and
spreads slowly upward. Initially, linear reddish-brown streaks appear, extending from the base to
the shoot tip. Lens-shaped cankers then develop. Shoots subsequently wilt, droop and dry up.
Discolouration is less common on very young shoots, but the whole shoot dies back. Where there
is severe infection, a large number of adventitious buds develop, but these quickly die back.
Infected shoots are shorter, giving the vine a stunted appearance. Tissue browning is revealed in
stem cross-sections. Infected grape bunch stalks show symptoms similar to the infected shoots.
Leaves may be penetrated via the petiole and then the veins, in which case the whole leaf dies.
Alternatively, leaves are penetrated directly via the stomata, resulting in the development of
angular, reddish-brown lesions. Infection through the hydathodes results in reddish-brown
discolourations in the leaf tips. Pale yellow bacterial ooze may be seen on infected leaves when
humidity is high.
Immature flowers turn black & die back.
Roots may also be attacked, resulting in retardation of shoot growth, regardless of grafted or
natural rootstock.
Symptom Images
Figure 1. Bacterial blight symptoms on shoots. Photo: C G Panagopoulis.
Figure 2. Bacterial blight infection on vine leaf. Photo: C G Panagopoulis.
Figure 3. Cankers on bunch stalks, causing partial or total fruit death. Photo: ARC- Nietvoorbu
South Africa.
Sites of Infection/Infestation
Bleeding sap appears to be an important source of contamination.
The disease can be transmitted by propagation material, during grafting and by pruning knives.
Bacteria are spread by moisture to plants where infection may occur through wounds, leaf scars
and other sites. Infection may also occur without wounds (Serfontein et al. 1997).
Factors Influencing Occurrence
The varieties Sultana and Barlinka are very susceptible. Hermitage is moderately susceptible and
Green Grape and Reisling are moderately resistant.
Identification
Isolation/Culture Techniques
Growth on artificial medium is very slow and a heavy inoculum is advisable as growth may fail
if streaks are made from dilute suspensions. On nutrient agar colonies are barely visible (0.2-0.3
mm diam) in 6 days at 26oC increasing to 0.6-0.8 mm in 15 days, and are circular, entire,
glistening, translucent, pale yellow. On nutrient agar with 5% sucrose or on GYCA (containing
1% glucose, 0.5% yeast extract, 3% calcium carbonate and 2% agar), growth is faster and deeper
yellow. Best growth is obtained on 2% galactose, 1% yeast extract, 2% calcium carbonate and
2% agar, on which it is deep yellow and produces a diffusible brown pigment. In minimal
synthetic medium 0.1% glutamate is required for growth, but L-methionine is not. No fluorescent
pigment is produced on King's Medium B or any similar medium.
No strain hydrolyses aesculin, arbutin, casein, gelatin, sodium hippurate or starch, or shows
pectolytic activity on potato tissue, reduces nitrate to nitrite, produces ammonia, indole, arginine
dihydrolase, ornithine and lysine decarboxylases or phenylalanine deaminase. H2S is produced
from cysteine and thiosulphate by all strains, but from peptone by some only. The malonate and
gluconate tests are negative, tyrosinase evidently varies. Lipolysis is positive on
Tween 80 but negative or weak on tributyrin. Urease is strongly positive; catalase positive;
oxidase negative.
Acid was produced without gas in 6-8 days from L-arabinose and D-galactose, but not from
glucose in Hugh & Leifson's medium with peptone reduced to 0.1%. Metabolism was strictly
aerobic. In Dye's Medium C with bromothymol blue indicator and 0.5% carbon source, acid was
produced from L-arabinose, D-galactose and slowly by some isolates from glycerol, but not from
adonitol, arbutin, cellobiose, dextrin, dulcitol, Dfructose, D-glucose, glycogen, meso-inositol,
inulin, lactose, maltose, D-mannitol, D-mannose, D-melibiose, amethyl-D-glucoside, raffinose,
L-rhamnose, D-ribose, salicin, D-sorbitol, L-sorbose, sucrose, trehalose or Dxlyose.
Some strains may produce acid from glucose (58, 4525) and Oleron strains are reported to
produce acid from maltose.
Citrate, fumarate, malate and tartrate are used as sole carbon sources, and succinate slowly, also
acetate at 0.2%, but not at 0.5%. Gluconate, maleate, malonate, oxalate, benzoate, formate and
propionate are not used.
The last 3 inhibit the small amount of growth seen on the minimal medium in the absence of a
carbon source.
Lactate is used by some strains.
Growth fails or is very poor in presence of 0.02% triphenyltetrazolium chloride. Max. tolerance
of NaC1 1%.
Min. temp. for growth 6oC, max. 30oC.
The G + C content of the DNA is 68 to 69 tool% by thermal denaturation. Type strain NCPPB
2217; ICMP 4298; ATCC 33914 (Bradbury, 1991).
Morphological Methods
X.ampelinus is a Gram-negative, non-spore forming rod with one polar flagellum, 0.5-0.8 x 1.63.2µ. Occasionally isolates have some filamentous cells 8 - 10 times longer than usual. Occurs
singly or in pairs. In culture, at 25oC, growth is slow, non-mucoid, smooth, yellow, round entire
colonies, 0.4-0.8 mm in diameter, develop in 6-10 days on yeast-glucose-chalk agar. On nutrient
agar with 5% sucrose and on sucrose-peptone-salts agar there is more growth and a deeper shade
of yellow. Growth is best on yeast extract 1%, galactose 2%, CaCO3 2% and agar 2% on which
medium it is deep yellow and produces a brown diffusible pigment.
Potentially misidentified species
Erwinia vitivora
Cane and leaf spot caused by the fungus Phomopsis viticola
Differs from Xanthomonas vitis-carnosae, X. vitis-trifoliae and most other members of the genus
in its very slow growth, low max. temp., low salt tolerance, inability to produce acid from
numerous sugars (including glucose), failure to hydrolyse gelatine, casein, starch and aesculin. It
most nearly resembles X. albilineans, but differs from this and other xanthomonads in its strong
urease activity and ability to use tartaric acid salts as carbon source.
Biological Methods
Standard bacteriological tests and three serological techniques for rapid identification of X.
ampelinus are described by Erasmus et al. (1974) - see below:
Erasmus, H.D., Matthee, F.N., Louw, H.A. (1974) A comparison between plant pathogenic
species of Pseudomonas, Xanthomonas and Erwinia with special reference to the bacterium
responsible for bacterial blight of vines. Phytophylactica 6: 11 - 18.
Another potentially useful reference is: Serfontein, S., Serfontein, J.J., Botha, W.J. (1997) The
isolation and characterisation of Xylophilus ampelinus. Vitis 36(4): 209 - 210.
The bacterium can be detected by immunofluorescent microscopy in washings from diseased
leaves or in homogenates of woody tissues, as well as in ooze or on pruning shears. The
bacterium may be founding stems and leaves up to 10 cm or more above visibly infected areas.
Molecular Methods
Botha, W.J., Serfontein, S., Greyling, M.M., Berger, D.K. (2001). Detection of Xylophilus
ampelinus in grapevine cuttings using a nested polymerase chain reaction. Plant Pathology
50(4), 515-526.
***** (ref. often quoted for detection) ********
Dreo, T., Gruden, K., Manceau, C., Janse, J.D., Ravnikar, M. (2007). Development of a real-time
PCR-based method for detection of Xylophilus ampelinus. Plant Pathology 56, 9-16.
López, M.M., Cambra, M., Aramburu, J.M., Bolinches, J. (1987) Problems of detecting
phytopathogenic bacteria by ELISA1. Bulletin OEPP/EPPO, Bulletin 17: 113 - 117.
Manceau, C., Grall, S., Brin, C., Guillaumes, J. (2005) Bacterial extraction from grapevine and
detection of Xylophilus ampelinus by a PCR and Microwell plate detection system. Bulletin
OEPP/EPPO Bulletin 35: 55 - 60.
Serfontein, S., Serfontein, J.J., Botha, W.J. (1997) The isolation and characterisation of
Xylophilus ampelinus. Vitis 36(4): 209 - 210.
Further Information
Contacts
If you have further information about this pest and would like to post it on this site, or would like
to amend or correct any information currently displayed, please contact:
Gary Kong
Principal Plant Pathologist
Plant Science, Delivery
PO Box 102 Toowoomba
Qld 4350
Ph: 61+7 4688 1319
mob: 61+ 428103521
Email: [email protected]
References
AQIS, 2002 Draft Review of Post Entry Quarantine Protocols for the Importation of Grapevine
(Vitis) into Australia. Department of Agriculture, Fisheries and Forestry.
Botha, W.J., Serfontein, S., Greyling, M.M., Berger, D.K. (2001). Detection of Xylophilus
ampelinus in grapevine cuttings using a nested polymerase chain reaction. Plant Pathology
50(4), 515-526.
Bradbury, J.F. (1973) Xanthomonas ampelina. CMI Descriptions of Pathogenic Fungi and
Bacteria No. 378. CAB International, Wallingford, UK.
http://www.cababstractsplus.org/DFB/Reviews.asp?action=display&openMenu=relatedItems&R
eviewID=9490&Year=1973
Bradbury, J.F. (1991) Xylophilus ampelinus. IMI Descriptions of Fungi and Bacteria
No.1050. Mycopathologia 115: 63 - 64.
Distribution Maps: CAB International, Nosworthy Way, Wallingford, Oxfordshire, OX10 8DE,
UK.
www.cabi.org/datapage.asp?iDocID=778
Dreo, T., Seljak, G., Janse, J.D., van der Beld, I., Tjou-Tam-Sin, L., Gorkink-Smits, P.,
Ravnikar, M. (2005) First laboratory confirmation of Xylophilus ampelinus in Slovenia. Bulletin
OEPP/EPPO Bulletin 35: 149 - 155.
Dreo, T., Gruden, K., Manceau, C., Janse, J.D., Ravnikar, M. (2007). Development of a real-time
PCR-based method for detection of Xylophilus ampelinus. Plant Pathology 56, 9-16.
EPPO/CABI, 1996. Viteus vitifoliae. In: Smith IM, McNamara DG, Scott PR, Holderness M,
eds. Quarantine Pests for Europe. 2nd edition. Wallingford, UK: CAB International.
EPPO Data Sheets on Quarantine Pests (undated) Xylophilus ampelinus.
http://www.eppo.org/QUARANTINE/bacteria/Xylophilus_ampelinus/XANTAM_ds.pdf
Erasmus, H.D., Matthee, F.N., Louw, H.A. (1974) A comparison between plant pathogenic
species of Pseudomonas, Xanthomonas and Erwinia with special reference to the bacterium
responsible for bacterial blight of vines. Phytophylactica 6: 11 - 18.
Fiori M, Carta C, Garau R, Prota VA, 1996. Further investigations on "mal nero" disease of
grapevine in Sardinia. Phytopathologia Mediterranea, 35:13-18.
Grodnitskya, I. D. and Gukasyan, A. B. (1999). Bacterial diseases of conifer seedlings in forest
nurseries in Central Siberia. Microbiology New York 68, 189-193.
Lopez MM, Gracia M, Sampayo M, 1981. Studies on Xanthomonas ampelina Panagopoulos in
Spain. Proceedings of the Fifth Congress of the Mediterranean Phytopathological Union, Patras,
Greece, 21-27 September 1980. Athens, Greece: Hellenic Phytopathological Society, 56-57.
López, M.M., Cambra, M., Aramburu, J.M., Bolinches, J. (1987) Problems of detecting
phytopathogenic bacteria by ELISA1. Bulletin OEPP/EPPO, Bulletin 17: 113 - 117.
Manceau, C., Grall, S., Brin, C., Guillaumes, J. (2005) Bacterial extraction from grapevine and
detection of Xylophilus ampelinus by a PCR and Microwell plate detection system. Bulletin
OEPP/EPPO Bulletin 35: 55 - 60.
Panagopoulos, C.G. (1987) Recent research progress on Xanthomonas ampelina. EPPO Bulletin,
17(2):225 - 230.
Ride, M., Ride, S. and Novoa, D. (1977). Donnes nouvelles sur la biologie de Xanthomonas
ampelina Panagopoulos, agent de la necrose bacterenne de la vigne. Annales de Phytopathologie
9, 87.
Serfontein, S., Serfontein, J.J., Botha, W.J. (1997) The isolation and characterisation of
Xylophilus ampelinus. Vitis 36(4): 209 - 210.
WA Agriculture Factsheet No. 0005-2000, date 05/2000 Bacterial blight, exotic threat to
Western Australia. Stansbury, C., McKirdy, S., Power, G.
http://www.agric.wa.gov.au/content/pw/ph/dis/vit/fs00500.PDF
Other useful websites:
http://www.plantprotection.hu/modulok/angol/grapes/blight%20_grap.htm