Characterisation of Xanthomonas campestris isolates that caused an outbreak
of a bacterial leaf spot disease of cabbage in Mauritius
K. Lobin, S.P. Benimadhu, D.Y. Dhooky (Agricultural Research Extension Unit, Mauritius)
Joseph Mulema, Vânia H. Passo, Eric B. Holub and Joana G. Vicente (School of Life Sciences, University of Warwick, UK)
Introduction
Cabbage (Brassica oleracea var. capitata) is a crop of economic importance grown in Mauritius by approximately 4000 growers producing 4500
tonnes annually on 200 hectares.
In January 2009, an outbreak of a leaf spot disease was observed in cabbage vars. Tropic King and Summer Power grown in the super humid
areas of La Marie, Mare Longue, Plaine Sophie and La Laura.
Overall Symptomatology
Isolation of the pathogen and Pathogenicity tests
Symptoms begin initially as small, brown necrotic spots, often with
chlorotic halos.
These expand and coalesce to cover large areas of the leaf surface.
Eventually cabbage heads get infected and unmarketable, causing
economic losses.
Colonies recovered from diseased tissues were
mucoid cream-yellow Xanthomonas-like and
white colonies on Nutrient Agar (NA).
Pathogenicity tests by spray inoculation of bacterial suspensions
on susceptible cabbage variety Tropic King showed that only the
Xanthomonas-like colonies are pathogenic.
After 14 days, inoculated plants developed leaf spots that were
identical to those observed in the field. Colonies with morphology
typical of Xanthomonas were recovered from leaf spots of
inoculated plants.
Molecular characterisation
3849A Xcc USA Race 2
16s and gyrase B sequencing
6382 Xcc Canada Race 5
Gyrase B phylogeny produced by using
the UPGMA and Jukes-Cantor method.
16s rDNA sequences of two isolates from Mauritius (HRI 8506 and 8514) confirmed
that the cream-yellow mucoid isolates are Xanthomonas spp..
5212 Xcc UK Race 3
6181 Xcc Portugal Race 6
8506 Xcc Mauritius
8450A Xcc Nepal Race 7
1279A Xcc UK Race 4
3811 Xcc USA Race 1
The gyrase B gene (1) was partially sequenced for isolate HRI 8506.
The results indicate that the isolate is an X. campestris pv. campestris (Xcc) with a
sequence identical to some other Xcc isolates including the race-type strain of race 4.
3880 Xcc Australia Race 5
6490 Xcr France
6518 Xcr France
8305 Xcr USA
5235 Xv
Rep-PCR fingerprinting
XopAH (AvrXccC)
Rep-PCR with REP, ERIC and BOX primers (2) was performed for three
isolates from Mauritius. In total, 36 polymorphic loci were scored.
The results show that the Mauritius isolates cluster with the Xcc isolates,
but are not identical to other tested Xcc isolates.
The avrXccC gene (3) was sequenced (length of approximately 1000bp)
for three isolates from Mauritius (HRI 8506, 8514, 8516A).
Two isolates have a mutation similar to the race-type strain of race 4.
8514 Mauritius
8506 Mauritius
Conserved
3849A USA
(from Ser to Gly)
6181 portugal
8450A Nepal
Rep-PCR phylogeny produced by using
the UPGMA and Jukes-Cantor method.
(Cophenetic correlation = 0.96)
1279A UK
5212 UK
3811 USA
8516A Mauritius
6382 Canada
avrXccC sequence (996bp)
Mutation at 685bp
Not present
5212 (race 3)
6181* (race 6)
8450A* (race 7)
1279A (race 4)
8506
8516A
3849A (race 2)
6382 (race 5)
8514
* Shorter sequences (824 and 975bp respectively)
Spray inoculated
Savoy cabbage
Wound inoculated
Arabidopsis Can-0
Pathogenic characterisation
One isolate (HRI 8506) was inoculated using wound and spray methods into eight Brassica hosts and twenty
Arabidopsis thaliana accessions.
The reaction on Brassica hosts was most similar to Xcc race 4 especially in wound inoculations; leaf spots were
observed in spray inoculations, but V-shape lesions typical of Xcc were also observed.
Spray inoculation caused more symptoms that wound inoculation in the A. thaliana accessions.
Resistant and susceptible accessions have been identified.
Conclusion
Based on molecular and pathological studies, the pathogen is most likely to be a variant of Xcc.
In extreme rainy and humid conditions that prevailed during the outbreak, it is possible that the pathogen has been able to infect cabbage leaves
through the stomata and cause leaf spots in contrast to the commonly known typical V-shaped symptoms of black rot disease reported on cabbage
in Mauritius. A similar outbreak has been reported on Brassicas in South Carolina, USA, with Xcc as causal organism (4).
Future work
The pathogenicity of several isolates will be tested in different Brassica spp. and Arabidopsis accessions.
Further molecular characterisation will include complete sequencing of one of the isolates.
Acknowledgement:
References:
We thank Dr Paul Hunter (Warwick HRI, University of Warwick, UK) for 16S sequences.
(1) Young et al. Syst. Appl. Microbiol, 31 (2008): 366-377; (2) Vicente et al. Phytopathol. 96 (2006): 735-745
(3) Wang et al. Mol. Plant Pathol., 8 (2007): 491-501
(4) Wechter et al. Plant Dis. 92 (2008): 1134