Detection of variability in xanthomonas campestris pv.

1. J. Appl. Hort., 2(1):44-46, January-June, 2000
Detection of variability in Xanthomonas campestris pv.
mangiFeraeindicae strains
R. Kishun and S. Rajan
Central Institute for Subtropical Horticulture, Rehmankhera, P. O. Kakori, Lucknow-227107, India.
E-mail: rkishun@rajan.com
Abstract
Nineteen Xanthomonas campestris pv. mangiferaeindicae strains collected from different ecogeographical areas/mango genotypes of
India were studied to confirm the existence of variability in the strains on the basis of their pathogenicity on genotypes (9), reaction
towards antibiotics (9) and growth on culture media (5). Study revealed the existence of variability in Xcmi strains as exhibited by their
differential reaction. The similarity in Xcmi strains was observed by hierarchical cluster analysis. Clustering pattern on these three
detection methods indicated that the grouping of strains is not entirely based on their geographical distribution as-the strains from
northern and southern parts of India falls in a single cluster. However, the strains collected from Bihar exhibited more similarity with each
other and clustered in one or nearby cluster in all the detection methods used.
Keywords: Mango, Mangifera indica, variability, Xanthomonas campestris pv. manqiferaeindicae, strains
Introduction strains except Xcmi 7 were spot inoculated (109
cfu Zrnl)in the month of September on single leaf of
each genotype and replicated 5 times. Data on size of
spots (mm) was recorded on 10th day after
appearance of initial disease symptoms.
Table 1. SourceofXanthomonas campestris pv. mangiferaeindicae
strains isolated from different mango growing;llfeas/cultivars of
India i
Mango (Mangifera indica L.) suffers from a number of
diseases caused by bacteria, fungi and other agents.
Since last few years, mango bacterial canker disease
(MBCD) incited by Xanthomonas campestris pv.
mangiferaeindicae (Xcmz) has become serious in India
and abroad (Kishun, 1995). The pathogen affects all
the above ground plant parts and causes heavy loss
to the crop under favourable environmental
conditions (Shekhawat and Patel, 1975; Kishun,
1981). Recently, it has been found to cause loss in
yield up to 70.61 per cent in cv. Dashehari (Kishun,
1997), considered tolerant to the disease. During
surveys for prevalence of MBCD in India, variation in
disease incidence and intensity were noticed
(Kishun, 1995). This variation in severity of MBCD
may be due to presence of variability in Xcmi strains.
Since, information on detection of such variation in
Xcmi in India is not available except some stray
reports based on limited strains. Therefore,
investigations were carried out in this direction and
results are reported.
Materials and methods"
Collection of bacterial strains: MBCD infected
plant samples were collected from different
agroclimatic regions/mango cultivars of India and
isolations were made on nutrient agar medium
(peptone-5g, beef extract-3g, D-glucose-5g, agar-15g
and distilled water-1000 ml). Cultures were purified
by single colony transfers, proved pathogenic and
designated (Table-I).
Varietal reaction: Mango grafts of cvs Amrapali,
Bombay Green, Chausa, Dashehari, Fajri, Malihabad
Safeda, Langra, Mallika and Ramkela were planted
in the field. After a year of their establishment all the
SI. Culture
No. designation
Source
1. Xcmi 1 Fruit Research Station, Sangareddy, A P.
2. Xcmi 2 Baiq's Nursery, Sangareddy, A P.
3. Xcmi 3 Anjaiah's Nursery, Sangareddy, AP.
4. Xcmi 4 Langra-I, CISH, Rehmankhera, Luc:know, U. P.
5. Xcmi 5 Langra-II, CISH, Rehmankhera, Lucknow, U. P.
6. Xcmi 6 Root Stock, CISH, Rehmankhera, Lucknow, U. P.
7. Xcmi 7 Progeny Orchard, Malihabad, Lucknow, U. P.
8. Xcmi 8 Farmer's Field, Bazpur'iU.P,
9. Xcmi9 Fruit Research Station, Basti, U. P.
10. Xcmi 10 IIHR, Bangalore, Karnataka
11. Xcmi 11 CHES, Ranchi, Bihar
12. Xcmi 12 Nursery, CISH, Rehmankhera, Lucknow, U. P.
13. Xcmi 13 Farmers' Field, Sabour, Bihar
14. Xcmi 14 Horticulture Garden, Sabour, Bihar
15. Xcmi 15 CHES, Ranchi-I, Bihar
16. Xcmi 16 CHES, Rancm-ll, Bihar
17. Xcmi 17 BAU,.Ranchi, Bihar
18. Xcmi 18 RAU, Pusa, Bihar
19. Xcmi 19 HRS, Virauli, Pusa, Bihar
Antibiotic sensitivity: In case of antibiotics, 19
Xcmi strains were evaluated against Ampicillin (10
mcg/ disc), Bacitracin (10 units / disc), Chlorarn-

2. Variability in Xanthomonas campestris pv. mangiferaeindicae strains
phenicol (30 mcg/disc), Erythromycin (15 mcg/disc},
Gentamicin (10 mcg/rlisc), Nystatin(100 mcg/rlisc],
Oxytetracycline (30 mcg/ disc), Penicillin G (30
units/disc) and Vancomycin (30 mcg/disc) supplied
by HIMEDIALaboratories Pvt. Ltd., India. Evaluation
was carried out on nutrient agar seeded with
bacterial suspension (109 cfu/ ml) of each Xcmi strain
separately in petriplates (100 mm). Antibiotic discs
were placed in the centre of seeded plates after
solidification and each antibiotic was replicated
thrice. The plates were incubated at 30 ± 1DCfor 48
hrs and data on inhibition zone (mm) was recorded
at the end of incubation period.
Growth on culture media: All the strains were
evaluated on various culture media (liquid) to find
out the differences in their quantitative growth. The
culture media tested were nutrient broth (NB),yeast
extract nutrient broth (YEN), yeast extract chalk
(YEC), King's B (KB)and casein glucose (CG)as per
their composition reported (Schaad and Stall, 1992).
Fixed quantity of pure cultures (20 ul] with
concentrations of 108 cfu/rnl of different strains were
inoculated separately in culture tubes of similar
shape and size and containing 5 ml sterile medium.
Data on growth was recorded after 72 hrs- of
incu bation at 30 ± 1DCwith Spectronic 20 at 460
nm.
Statistical analysis: Data obtained on the reaction
of Xcmi strains with genotypes, antibiotics and
culture media, were subjected to hierarchical
analysis for classifying the strains. Euclideon
distance and Ward's minimum variance technique
(Romesburg, 1984) was used for clustering the
strains.
Results
The Xcmi strains were clustered separately on the
basis of their reaction with genotypes, antibiotic and
culture media. Data presented in dendogram (Fig.
IB) clearly show that the strains varied in their
pathological reactions on mango genotypes. Based on
the clustering pattern with genotypes the strains can
be clustered in 4 major groups. These are Xcmi 8, 10,
6 and 9 (A);Xcmi 4 and 5 (B);Xcmi 14, 16, 15, 11, 12
and 13 (C) and Xcmi 17, 18, 19, 3, 1 and 2 (D).
Similarly the strains (19) showed variation in their
reaction towards antibiotics and categorized in 4
groups (Fig. lA). These groups are Xcmi 2,7, 12, 1,
4, 9, 5, 8 and 10 (A);Xcmi 11 (B);Xcmi 3,6 and 14
(C) and Xcmi 13, 17, 15, 19, 16 and 18 (D). The
quantitative growth of Xcmi strains was also varied
on tested culture media (Fig. le) and clustered in 4
closely related strains group. These are Xcmi 9, 11,
8,5,14,18,19,1,15 and 16 (A);Xcmi 2,3 and 17
(B); Xcmi 7 and 12 (e) and Xcmi 4,13,6 and 10 (D).
Here it is interesting to note that all the
representative strains of group D (antibiotic
sensitivity) were collected from Bihar, while the
group C formed on the basis of genotype reaction
had all the strains from Bihar except Xcmi 12. The
pattern exhibited in group A of Fig. 1C (culture
45
media) depicts that most of the strains belong to
Bihar. Thus it is clear from the results that the
strains collected from different geographical
areas / genotypes were not similar except from Bihar,
i.e., Xcmi 11,14,15,16,17,18 and 19 which falls
almost in same cluster in all the detection methods.
o
I
5
Rescaled Distance
10 15 20 25
Xcmi
strains
2
7
12
1
A 4
9---J
5
8
10
B J:11 -------'
cl!14---'
o I~~19
16_..1-_ ....•
18---l
1A
11~
A 6
9
B I ~
c III12
13
D I:l--r-~---------------J
18
,
,
9
11
8
5-....-- ....•
A 14
18-....-----'
19
1 -r-..,
15 ~-----~
16-- .....•
B I~-------'17 -J
cl 7
12
o I1~ -,--..,
10-------- ....•
1C
Fig. 1. Dendrogram showing groups in Xanthomonas campestris
pv. mangiferacindicae.strains based on their reaction towards
mango genotypes (8), antibiotic sensitivity (A) and growth on
culture media (C).

3. Discussion
Journal of Applied Horticulture46
Variations among the Xcmi strains (19) collected from
different agroclimatic regions/mango genotypes was
detected by their pathological reactions on mango
cvs, sensitivity towards antibiotics and growth on
culture media. But the same group of strains have
not been observed in one cluster in all the detection
methods. However, some of them such as Xcmi 15
and 16 were found in a single cluster in all the
testing methods, while some others (Xcmi 8 and 10
and 17, 18 and 19) were in one cluster in two
detection methods. It means the variability in strains
is not entirely based on ecogeographical distribution
except strains from Bihar (Xcmi 11, 13, 14, 15, 16,
17, 18 and 19) which were either in same or nearby
cluster. Such geographical specificity has not been
reported in Xcmi but known to occur in citrus
bacterial spot (Hartung and Civerolo, 1991) and
sugarcane scald (Rott et al. 1986; Yang et al. 1993)
pathogens. In some of the regions strains exhibited
diverse reaction which indicate that the evolutionary
trend of strains are not only geographically
dependent but also related with other factors
possibly host types. This can be seen by the diverse
behaviour of two strains from Lucknow where one
was isolated from local variety Langra (Xcmi 5) and
other from south Indian polyembryonic variety Goa
(Xcmi 6). Probably, another cause for diverse reaction
may be exchange of germplasm through which
strains of one region might have traveled distantly
distributed mango growing areas. In general, all the
three detection methods, i.e., reaction towards
mango genotypes, antibiotics and culture media gave
diverse reactions to Xcmi strains, indicating that the
degree of genetic similarity may not be so explicitly
depicted by the methods used to differentiate the
diversity among the Xcmi strains. Wachters et al.
(1991) had tested mango genotypes for pathogenicity
difference in Xcmi but they did not found any
variation. Presence of groups in Xcmi strains on the
basis of cultural, biochemical and pathological
characters are reported by Venugopal et al. (1991)
and Dayakar and Gnanamanickam (1996) but the
strains tested by them were mostly collected from
southern India. Groups in Xcmi on the basis of
phenotypic, i.e., isoenzyme (Some and Samison,
1996) and carbohydrate utilization profile (Pruvost et
al., 1998) and genomic (Gagnevin et al., 1997)
diversities have been reported from abroad. Though
different workers have tried various methods for
clustering in Xcmi but they have not used
hierarchical method of analysis except in case of
genomic diversity where PROC CLUSTER procedure
was used by Adhikari et al. (1995). It emanates from
the present findings that the variability in Xcmi can
be detected by pathological reaction on mango
cultivars, antibiotic sensitivity and growth on culture
media and hierarchical analysis may be used for
clustering the strains.
Acknowledgement
Authors are grateful to Dr. S. S. Negi, Director, CISH,
Lucknow for providing necessary facilities and
encouragement during the course of investigation.
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