Morphological, pathological and molecular variability in Botryodiplodia theobromae (Botryosphaeriaceae) isolates associated with die-back and bark canker of pear trees in Punjab, India

Abstract

Thirteen isolates of Botryodiplodia theobromae collected from pear varieties grown in various regions of Punjab were studied for morphological, pathological and molecular characterization. The mycelial growth of B. theobromae isolates was classified as fluffy or depressed, uniform to irregular and cottony white turning to black. Colony growth rate varied from 19.1 to 24.9 mm per day. Pycnidia were produced either on the edge, centered or scattered on Petri dishes after 20 to 34 days of incubation. Pycnidia and pycnidiospores ranged in size from 118.0 to 240.0 microm and 14.5-35.5 x 6.5-14.5 microm, respectively. Lesion length produced by different isolates ranged from 1.9-7.2 x 0.8-3.3 cm with 49.4-90.9% infection. Using nine SSR and seven RAPD markers, amplified DNA bands ranged from 0.2 to 1.5 and 0.18 to 2.0 kb, respectively. Polymorphism information content values ranged from 0.44 to 0.71 and 0.63 to 0.93 for SSR and RAPD markers, respectively. A dendrogram based on molecular data, grouped the isolates into three major clusters with 65 to 79.5% genetic similarity; most of the isolates showed variety-specific grouping. The isolates prevalent on pear cultivars 'Patharnakh' and 'Baggugosha' in Ludhiana, Amritsar and Hoshiarpur districts were found to have a high degree of similarity; these isolates were also considerably distant from mango isolates and from other isolates on other pear cultivars. The isolates from cultivars Punjab Beauty, LeConte and Kieffer also had a high degree of similarity. Isolates from cultivar Smith were different from other pear isolates but showed more similarity with mango isolates.

Full text

©FUNPEC-RP www.funpecrp.com.br
Genetics and Molecular Research 9 (2): 1217-1228 (2010)
Morphological, pathological and molecular
variability in Botryodiplodia theobromae
(Botryosphaeriaceae) isolates associated
with die-back and bark canker
of pear trees in Punjab, India
M.D. Shah1, K.S. Verma2, K. Singh3 and R. Kaur2
1Division of Plant Pathology,
Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir,
Shalimar, Srinagar, Jammu and Kashmir, India
2Department of Plant Pathology, Punjab Agricultural University, Ludhiana,
Punjab, India
3School of Biotechnology, Punjab Agricultural University, Ludhiana, Punjab,
India
Corresponding author: M.D. Shah
E-mail: mehraj547@rediffmail.com
Genet. Mol. Res. 9 (2): 1217-1228 (2010)
Received March 4, 2010
Accepted April 3, 2010
Published June 29, 2010
DOI 10.4238/vol9-2gmr812
ABSTRACT. Thirteen isolates of Botryodiplodia theobromae collected
from pear varieties grown in various regions of Punjab were studied
for morphological, pathological and molecular characterization. The
mycelial growth of B. theobromae isolates was classied as uffy or
depressed, uniform to irregular and cottony white turning to black.
Colony growth rate varied from 19.1 to 24.9 mm per day. Pycnidia were
produced either on the edge, centered or scattered on Petri dishes after
20 to 34 days of incubation. Pycnidia and pycnidiospores ranged in
size from 118.0 to 240.0 µm and 14.5-35.5 x 6.5-14.5 µm, respectively.
Lesion length produced by different isolates ranged from 1.9-7.2 x 0.8-

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3.3 cm with 49.4-90.9% infection. Using nine SSR and seven RAPD
markers, amplied DNA bands ranged from 0.2 to 1.5 and 0.18 to 2.0
kb, respectively. Polymorphism information content values ranged from
0.44 to 0.71 and 0.63 to 0.93 for SSR and RAPD markers, respectively.
A dendrogram based on molecular data, grouped the isolates into
three major clusters with 65 to 79.5% genetic similarity; most of the
isolates showed variety-specic grouping. The isolates prevalent on
pear cultivars ‘Patharnakh’ and ‘Baggugosha’ in Ludhiana, Amritsar
and Hoshiarpur districts were found to have a high degree of similarity;
these isolates were also considerably distant from mango isolates and
from other isolates on other pear cultivars. The isolates from cultivars
Punjab Beauty, LeConte and Kieffer also had a high degree of similarity.
Isolates from cultivar Smith were different from other pear isolates but
showed more similarity with mango isolates.
Key words: Botryodiplodia theobromae; Molecular markers; Pear;
Punjab; Variability
INTRODUCTION
The fungal pathogen Botryodiplodia theobromae Patouillard [Lasiodiplodia theobro-
mae (Patouillard) Griffon and Maublanc] represents the sexual state of Botryosphaeria rhodina
(Berk. and M.A. Curtis) Arx. It is an important opportunistic pathogen with worldwide distribu-
tion in tropical and subtropical regions causing different types of diseases in many plant species.
It is one of the economically important fungi that attack a wide range of hosts (Punithalingam,
1976). B. theobromae causes shoot blight, die-back, twig blight, cankers, etc., mainly in woody
plants including fruit and tree crops such as pear, apple, Albizia falcataria, peach, mango, avo-
cado, Citrus spp, Eucalyptus spp, Azadirachta indica, Pinus spp, etc. (Sharma et al., 1984;
Verma and Cheema, 1984; Mattos and Ames, 1986; Sharma and Sankaran, 1987; Darvas and
Kotze, 1987; Britton et al., 1990; Sangchote, 1991; Cedeno and Palacios-Pru, 1992; Cedeno
et al., 1995; Mohali et al., 2005). Die-back and bark canker is extremely destructive in certain
areas where climatic conditions are favorable for its development. The disease is conned to
the current growth and 1- to 2-year-old twigs. The bark of infected parts shrinks considerably
resulting in depressed lesions. Longitudinal and transverse cracks appear on the cankered bark
of some older branches (Verma and Cheema, 1984). The pathogen causes distension, disrupts
the cell walls and weakens the strength and toughness of wood (Shah, 2007).
DNA-based markers have been used to determine population genetic structures, gene
ow and reproductive mode in many fungal pathogens such as Botryosphaeria spp and their
anamorphs (Barnes et al., 2001; Zhou et al., 2002; Burgess et al., 2003; Slippers et al., 2004).
Simple sequence repeat (SSR) markers represent a class of co-dominant molecular markers
consisting of tandem repeat loci, rich in polymorphism with allele size determined by the addi-
tion or deletion of one or more repeats (Mohali et al., 2005). The molecular biology techniques
are now used in conjunction with morphological and physiological markers for the analysis
of populations. Few attempts have been made to organize B. theobromae isolates into groups
on the basis of genetic characteristics. Mohali et al. (2005) studied genetic diversity and gene

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ow between the populations of L. theobromae in Australia and observed that a small num-
ber of genotypes in Venezuela and South Africa populations showed low genotypic diversity.
However, the genotypic diversity was very high due to very high gene ow between the popu-
lations from different hosts. Burgess et al. (2003) collected B. rhodina isolates from pines and
Eucalyptus from South America, Mexico and Indonesia and found that 8 pairs were polymor-
phic among the isolates collected from Pinus spp. A further 5 pairs were polymorphic when
isolates from Pinus spp were compared with those from Eucalyptus spp thereby revealing
relationships among isolates to be more closely linked to the host than the geographical origin.
Botryodiplodia theobromae is one of the important pathogens in pear (Prunus spp)-
growing areas of the Punjab State in India. Analysis of variability in this pathogen is of great
signicance to identify the race spectrum of the pathogen and no study has been carried out
in India so far. Therefore, the aim of the present study was to evaluate the morphological
and pathological variability in B. theobromae isolates collected from different pear-growing
regions of the Punjab State, as well as their molecular characterization using SSR and random
amplied polymorphic DNA (RAPD) markers.
MATERIAL AND METHODS
Source of Botryodiplodia theobromae cultures
Thirteen isolates of B. theobromae were collected from commercial pear-growing re-
gions of the Punjab State. B-PL1, B-PL2, B-PL3, B-PL4, B-PL5, B-PL6, and B-ML were isolated
from pear cultivars Patharnakh, Baggugosha, Punjab Beauty, Kiefer, Smith, LeConte, and man-
go cultivar Dashehri, respectively, in the district of Ludhiana. B-PA1, B-PA2 and B-PA3 were
isolated from pear cultivars Patharnakh, Baggugosha and Punjab Beauty, respectively, in the
district of Amritsar, and B-PH1, B-PH2 and B-MH were isolated from pear cultivars Patharnakh,
Baggugosha and mango cultivar Dashehri, respectively, in the district of Hoshiarpur (Table 1).
Each isolate was maintained as a pure culture on potato dextrose agar (PDA) and stored at 4°C.
The stock culture of each isolate was revived on PDA by placing pycnidia on PDA with the help
of an inoculating loop. Petri dishes were incubated in a B.O.D. incubator at 30 ± 1°C.
Table 1. Source of Botryodiplodia theobromae isolates from pear growing regions of Punjab.
S. No. Isolate number Place of collection Pear variety
Ludhiana District
1 B-PL1 New orchard, PAU Patharnakh
2 B-PL3 Punjab Beauty
3 B-PL2 Old orchard, PAU Baggugosha
4 B-PL4 Kiefer
5 B-PL5 Smith
6 B-PL6 LeConte
7 B-ML Mango cultivar Dashehri
Amritsar District
8 B-PA1 Manawala Patharnakh
9 B-PA2 Baggugosha
10 B-PA3 Punjab Beauty
Hoshiarpur District
11 B-PH1 Hoshiarpur Patharnakh
12 B-PH2 Baggugosha
13 B-MH Mango cultivar Dashehri
PAU = Punjab Agricultural University.

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Morphological characterization of isolates
Isolates of B. theobromae were sub-cultured on PDA medium on Petri dishes (90 mm
in diameter) for comparison of morphological and growth characters. Eight-millimeter discs
from margins of actively growing 7-day-old fungal culture were inoculated on the center of
Petri dishes containing PDA medium. Three plates were used for each isolate. Colony growth
rate was recorded by measuring colony diameter after 24, 48, 72, 96, 120, and 144 h. Colony
color and texture observations were recorded after 7 days of incubation.
Pycnidial characters of each isolate viz. pycnidial number, arrangement, size, and
color were observed after 30 days of incubation. Pycnidiospore size, color and septation were
also recorded using a compound microscope.
Pathogenic behavior of isolates
The detached twig inoculation technique was used for studying the pathogenic behav-
ior of B. theobromae isolates on pear cultivar Baggugosha. A moist chamber was prepared by
placing a blotter sheet on a plastic tray (45 x 35 x 7.5 cm). Pear twig pieces of 6-8 cm long and
approximately 2 cm thick of similar age were taken from healthy branches, washed twice with
distilled water, air dried on sterilized blotting paper, and again surface sterilized with absolute
alcohol. An injury was inicted on the surface of twigs with the help of a cork borer. Inocula-
tion was done by placing 15-day-old 8-mm culture discs of each isolate in an inverted position
both on injured and uninjured sites of inoculation. A check was maintained in a similar manner
on twigs, which were not inoculated. Five twigs were used for each isolate. The trays with
twigs were incubated in a growth chamber (at 30 ± 1°C, on a 16-h light cycle). The observa-
tions were recorded regularly for symptom development.
Molecular characterization of isolates
Isolation of genomic DNA
A mycelial culture of each isolate was grown on potato dextrose broth. The mycelium of
15-day-old culture was dried and ground to a ne powder in liquid nitrogen using pre-cooled pes-
tle and mortar. Total genomic DNA of individual B. theobromae was extracted using the CTAB
method (Murray and Thompson, 1980). Isolated DNA was puried by using 1 µL RNase A (50
µg/mL) and resolved on 0.8% agarose gel. A working DNA solution was made by diluting the
DNA stock to 25 ng/µL with distilled water for polymerase chain reaction (PCR) amplication.
PCR amplication
SSR and RAPD primers (Table 5) were used for ngerprinting of B. theobromae iso-
lates. In preliminary experiments, 9 SSR primers were screened with one isolate of B. theobro-
mae and the primers depicting the best amplication were selected and used for SSR analysis
of all the isolates of B. theobromae. PCR was performed in 0.2-mL PCR tubes with a 20-µL
reaction volume contain ing 2.0 µL 10X buffer (10 mM Tris-HCl, pH 8.0, 50 mM KCl and
1.5 mM MgCl2), 4.0 µL dNTP mix (10 mM; Bangalore Genei, India), 1.6 µL primers (0.2

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µM: 0.8 µL of each forward and reverse), 0.3 µL Taq polymerase (Bangalore Genei; 5 U/µL),
2.5 µL DNA template (25 ng/µL) and 9.6 µL sterilized distilled water. The reaction mixture
was vortexed and centrifuged briey in a micro-centrifuge (Bangalore Genei). Amplications
were performed using a thermal cycler (MJ Research PTC200 or Eppendorf Master Cycler)
programmed for initial denaturation at 95°C for 2 min followed by 35 cycles at 95°C for 30 s,
annealing of primers at 52-68°C (depending on the primer used) for 40 s, extension at 72°C for
45 s (10 cycles) and for 50 s (25 cycles) and a nal extension at 72°C for 10 min using fastest
ramp time between the temperature transitions. The amplied PCR products were resolved by
electro phoresis using 1.5% agarose gel in 0.5X Tris borate EDTA buffer (0.5 M Tris, 0.05 M
boric acid and 1 mM EDTA, pH 8.0). DNA ladders of 100 bp (Bangalore Genei) and Lambda
DNA/EcoRI-HindIII-double digest (MBI Fermentas) were used as markers. The gels were
run at 5 V/cm for 2-3 h using a Bangalore Genei power system, stained with ethidium bro-
mide (0.5 µg/mL), and visualized by images captured using a UVP gel documentation system
(FOTODYNE) with the Foto/Analyst® PC Image software.
After screening 18 RAPD primers, 7 were selected for further analysis of all isolates.
For RAPD analysis, a 20-µL PCR volume containing 2.0 µL 10X buffer (10 mM Tris-HCl,
pH 8.0, 50 mM KCl and 1.5 mM MgCl2), 4.0 µL dNTP mix (10 mM; Bangalore Genei), 6 µL
primers (50 ng/µL), 0.3 µL Taq polymerase (Bangalore Genei; 5 U/µL), 2 µL DNA template
(25 ng/µL) and 5.7 µL sterilized distilled water was used. The thermal cycling proles for
RAPD were denaturation of genomic DNA at 94°C for 5 min, primer annealing at 37°C for
1 min and extension of primer at 72°C for 2 min. Forty additional PCR cycles were carried
out at 94°C for 1 min, 37°C for 1 min and 72°C for 2 min. An additional cycle at 72°C for 5
min was run at the end of these cycles. The PCR products were resolved by electrophoresis on
1.5% agarose gel in 0.5X TBE buffer at 50 V for 3 h.
Data analysis
DNA ngerprint data generated by SSR and RAPD markers were converted into bi-
nary matrix data and analyzed by the DICE coefcient and the UPGMA method with the
NTSYS 2.0 pc software (Rohlf, 1997) and a dendrogram was constructed.
RESULTS
Morphological variability among isolates
Of the 13 isolates studied for their morphological characters, 6 isolates, namely B-PA2,
B-PH1, B-PA1, B-PL1, B-PL2, and B-PH2, showed a colony growth rate of 24.0 to 24.9 mm/day
indicating their fast growing nature (Table 2). These isolates lled the entire 90-mm diameter
Petri dish surface in 96 h. Six isolates, i.e., B-PL3, B-PL5, B-PA3, B-PL4, B-PL6, and B-MH, ex-
hibited a moderate growth rate that ranged between 21.6 and 23.5 mm. The lowest growth rate
was observed by the isolate B-ML. The majority of the isolates (8) showed vegetative growth,
3 showed uffy colony growth and 2 isolates were appressed to the culture medium. Almost all
the isolates showed a uniform growth pattern, except B-PL4 and B-PL6, which exhibited an ir-
regular growth pattern. The colony color of isolates was dull white (B-PA1, B-PH2, B-PL3, and
B-MH), cottony white (B-PL5), cottony white changing to black (B-PL4, B-PL6 and B-PH1),

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cottony greyish black (B-PL1 and B-PL2), greyish white (B-PL1 and B-PL2) and whitish (B-PL6).
The isolates varied considerably with respect to pycnidial and pycnidiospore charac-
ters and 2 isolates (B-PL5 and B-PL6) did not sporulate on culture medium (Table 3). Time
taken for pycnidial production varied from 20 to 34 days under articial conditions. Minimum
time for pycnidial production was observed for the isolate B-PL2 (20 days) and maximum
time for the isolate B-ML (34 days). Pycnidial arrangement was on the periphery in 6 isolates,
scattered in 3 and centered in 2. Pycnidial diameter in isolates varied from 118 to 240 µm.
The pycnidiospore size varied from 14.5-35.5 x 6.5-14.5 µm and was maximum in the isolate
B-PL1
and minimum in B-PL4 and B-PL3 isolates.
Table 2. Colony characters of Botryodiplodia theobromae isolates collected from different pear growing regions
of Punjab.
Isolates Colony* growth rate (mm/day**) Sporulation# Colony texture and color
B-PL1 24.3 + Raised, uniform, cottony to greyish black
B-PL2 24.0 + Fluffy, uniform, cottony to greyish black
B-PL3 23.5 + Appressed, uniform, dull white
B-PL4 22.1 + Raised, irregular, cottony white changing to black
B-PL5 23.2 - Fluffy, uniform, cottony white
B-PL6 20.8 - Appressed, irregular, whitish in color
B-ML 19.1 + Raised, uniform, cottony white changing to black
B-PA1 24.7 + Raised, uniform, dull white
B-PA2 24.9 + Raised, uniform, greyish white in color
B-PA3 23.2 + Fluffy, uniform, greyish white in color
B-PH1 24.8 + Raised, uniform, cottony white changing to black
B-PH2 24.3 + Raised, uniform, dull white in color
B-MH 21.6 + Raised, uniform, dull white
CD (P = 0.05) 0.9
*Mean of three replications; **24 h (based on growth at 96 h); #Sporulation after 35 days of incubation (+ = observed;
- = not observed); B = Botryodiplodia theobromae; P = pear; M = mango; L = Ludhiana; A = Amritsar; H = Hoshiarpur;
1 = Patharnakh; 2 = Baggugosha; 3 = Punjab Beauty; 4 = Kieffer; 5 = Smith; 6 = LeConte. CD = critical difference.
Table 3. Pycnidial and pycnidiospore characters of Botryodiplodia theobromae isolates collected from different
pear growing regions of Punjab.
Isolates Pycnidial characters* Pycnidiospore size (µm)
Time taken (days) Diameter (µm) Arrangement
B-PL1 22 142-228 Periphery 27.0-35.5 x 10.0 -14.5
B-PL2 20 122-239 Periphery 21.5-32.0 x 11.5-13.5
B-PL3 29 126-230 Centered 8.0-23.0 x 6.5-9.0
B-PL4 31 131-209 Centered 14.5-21.0 x 7.0-8.5
B-ML 34 124-179 Scattered 15.5-22.0 x 9.0-13.5
B-PA1 24 118-240 Periphery 22.5-27.0 x 11.5-13.0
B-PA2 23 122-238 Periphery 24.5-25.0 x 10.7-8.0
B-PA3 28 127-218 Scattered 19.0-22.5 x 11.0-13.5
B-PH1 22 131-235 Periphery 24.0-26.0 x 6.5-9.0
B-PH2 21 126-237 Periphery 20.5-22.5 x 7.5-12.5
B-MH 33 125-189 Scattered 17.2-20.5 x 8.5-9.5
CD (P = 0.05) 4.3 6.8
*Mean of three observations after 35 days of incubation at 30 ± 1°C; B = Botryodiplodia theobromae; P = pear; M =
mango; L = Ludhiana; A = Amritsar, H = Hoshiarpur; 1 = Patharnakh; 2 = Baggugosha; 3 = Punjab Beauty; 4 = Kieffer; 5
= Smith; 6 = LeConte. CD = critical difference.

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Pathological variability among isolates
Morphologically similar B. theobromae isolates responded variably with respect to
the pathogenicity test on pear cultivar ‘Baggugosha’ under controlled conditions (Table 4).
The least similar relationship was observed between morphological traits of the isolates and
their pathogenic behavior. The isolates were able to infect the injured bark surfaces only, and
no infection was observed in uninjured bark. Isolates B-ML and B-MH were able to infect
mango cultivar Dashehri only. However, all the isolates from pear were able to infect both pear
and mango hosts. The symptoms developed on healthy twigs after 3-7 days of inoculation. The
percent infection in twigs by different isolates was in the range of 49.4-90.9% with a mean
of 77.5%. It was the highest (90.9%) in isolates B-PL1 and B-MH and the lowest (49.4%) in
isolate B-PL5. The average lesion size was in the range of 1.9-7.2 x 0.8-3.3 cm with an overall
mean of 5.5 x 1.9 cm in different isolates. The largest lesion size (7.2 x 2.3 cm) was noticed in
isolate B-PL1 and the smallest (1.9 x 0.8 cm) in isolate B-PL6.
DNA ngerprinting of Botryodiplodia theobromae
Genetic diversity was detected in 13 B. theobromae isolates by SSR and RAPD
markers. On the basis of the amplication produced, 9 SSR and 7 (of 18) RAPD primers
were screened to study the genetic variability. For genetic analysis of 13 B. theobromae
isolates, SSR and RAPD primers produced multiple PCR products. A range of 2 to 4 bands
with a total of 27 bands of 0.2 to 1.5 kb were obtained mainly with SSR primers. The banding
pattern obtained by the primer LAS15and16, with all isolates, is depicted in Figure 1a. This
primer produced 4 polymorphic bands in different isolates in the range of 0.2-0.6 kb. The
primer BOT17and18 produced 3 polymorphic bands in different isolates in the range of 0.3-
0.9 kb, indicating diversity among all the isolates (Figure 1b). The PCR products of 0.2-0.5,
Table 4. Pathogenic behavior of Botryodiplodia theobromae isolates collected from different pear growing areas
of Punjab following detached twig method on pear cultivar Baggugosha.
Isolates Incubation period (days) Percent of infection Average lesion size (cm)*
B-PL1 3 90.9 7.2 x 2.3
B-PL2 3 82.8 7.1 x 2.4
B-PL3 4 65.6 4.2 x 1.3
B-PL4 6 69.7 3.6 x 1.4
B-PL5 7 49.4 2.9 x 1.0
B-PL6 6 62.7 1.9 x 0.8
B-ML 7 82.5 6.3 x 1.9
B-PA1 4 89.3 7.0 x 2.3
B-PA2 4 81.8 6.8 x 2.5
B-PA3 5 72.7 5.1 x 1.5
B-PH1 3 87.5 6.5 x 2.8
B-PH2 5 81.8 6.2 x 1.4
B-MH 4 90.9 6.2 x 3.3
Mean 4.7 77.5 5.5 x 1.9
CD (P = 0.05) - 6.8 -
*After 15 days of incubation. Values are means of three replications. B = Botryodiplodia theobromae; P = pear;
M = mango; L = Ludhiana; A = Amritsar; H = Hoshiarpur; 1 = Patharnakh; 2 = Baggugosha; 3= Punjab Beauty;
4 = Kieffer; 5 = Smith; 6 = LeConte. CD = critical difference.

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specically present in isolates B-PL1, B-PL2, B-PL8, B-PL9, B-PL11, and B-PL12, were collected
from different districts of the Punjab State. The PCR products of 0.6-1.5 were specically
present in isolates B-PL3 and B-ML13. However, isolates B-PL4, B-PL5, B-PL6, B-PA3, and
B-ML7 presented banding patterns of varying size with different primers used.
Figure 1. Ethidium bromide-stained DNA amplication prole of 13 isolates of Botryodiplodia theobromae
using (a and b) microsatellite (SSR) and (c and d) randomly amplied polymorphic DNA (RAPD) markers. a.
LAS15and16. b. BOT17and18. c. S1120. d. S1118. M = DNA marker; C = negative control; lane 1 = B-PL1;
lane 2 = B-PL2; lane 3 = B-PL3; lane 4 = B-PL4; lane 5 = B-PL5; lane 6 = B-PL6; lane 7 = B-ML; lane 8 = B-PA1;
lane 9 = B-PA2; lane 10 = B-PA3; lane 11 = B-PH1; lane 12 = B-PH2; lane 13 = B-MH.

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Morphological, pathological and molecular variability in B. theobromae
Of the 18 RAPD primers used in the preliminary analysis, 7 were selected for genetic
analysis of 13 B. theobromae isolates on the basis of good amplication. Seven RAPD primers
produced 3-14 bands with a total of 51 bands. The primer S1120 yielded 7 bands in the range of
0.18-2.0 kb showing polymorphism among all the 13 isolates. The primer S1118 also produced
7 bands in the range of 0.5-2.4 kb, showing polymorphic DNA banding pattern. The primer
S111 followed by S1110 produced the highest number of bands (14 and 10, respectively).
Polymorphic information content values
The polymorphic information content (PIC) values provide an estimate of the discrim-
inatory power of a locus by taking into account not only the number of alleles that are detected
but also the relative frequencies of these alleles (Table 5). The PIC values for SSR markers
ranged from 0.44 for marker LAS27and28 to 0.71 for marker BOT19and20 with an average of
0.57 for all 9 markers. The PIC values of RAPD markers ranged from 0.63 in marker S1104 to
0.93 in marker S111 with an average of 0.81 for all 7 markers. Marker S111 amplied a total
of 14 loci in 13 isolates of B. theobromae with a PIC value of 0.93, whereas marker S1110
amplied only 10 alleles with a PIC value of 0.88. Thus, in the present set of genotypes, SSR
marker BOT19and20 followed by marker LAS15and16 and RAPD marker followed by marker
S1110 were the most informative as compared to all other selected markers.
Primer Primer sequence (5’ to 3’) Annealing Total No. Polymorphic Percent PIC
temperature (°C) of bands bands (No.) of polymorphism values
SSR
LAS13and14 F: 5’-GAGTTGTTAGTGCGGGCGCC-3’ 58 3 2 67 0.59
R: 5’-GCAGCCCCACAATTCACCAG-3’
LAS15and16 F: 5’-GCCAGATCCGTGCCCACTG-3’ 58 4 4 100 0.65
R: 5’-CATGCAGAGGTCGCAAAGTG-3’
LAS21and22 F: 5’-GGAAGATGATGGGATGGTTGC-3’ 55 2 1 50 0.50
R: 5’-GTACAAGAACGAACTCCGGGT-3’
LAS27and28 F: 5’-CGAACAGGGTTTCGTGACGT-3’ 52 2 1 50 0.44
R: 5’-CTCATATCTCGCCGGTTGCC-3’
LAS35and36 F: 5’-GGCATCACAACGACCAACCC-3’ 58 2 1 50 0.48
R: 5’-GCGAGAGTCGCAAGTACAGC-3’
BOT11and12 F: 5’-CGGCATGGTCTGCCGCTCC-3’ 68 3 2 67 0.61
R: 5’-GCATCTCCGGCTACCAACCG-3’
BOT17and18 F: 5’-GGCGCAATCTCGATTCGAGC-3’ 57 3 1 33 0.62
R: 5’-CCACGATGTCCGTTCATCG-3’
BOT19and20 F: 5’-GGCGGTCGCAGATGCGGTC-3’ 62 4 4 100 0.71
R: 5’-GCCCTATTCTGCGTGCCTCC-3’
BOT35and36 F: 5’-CTCCATCCTGATCCAGGGTCC-3’ 65 4 3 75 0.50
R: 5’-GACGAATCAAGCGGGCTGCCC-3’
Total 27 5.10 (0.57)*
RAPD
S111 5’-CTTTCCGCAGT-3’ 37 14 14 100 0.93
S116 5’-TCTCAGCTGG-3’ 37 5 4 80 0.77
S1104 5’-GAGGGACCTC-3’ 37 3 2 67 0.63
S1109 5’-ACCACGAGTG-3’ 37 5 3 60 0.75
S1110 5’-CAGACCGACC-3’ 37 10 10 100 0.88
S1118 5’-ACGGGACTCT-3’ 37 7 6 86 0.85
S1120 5’-ACCAACCAGG-3’ 37 7 7 100 0.85
Total 51 5.66 (8.10)*
*Average. SSR = simple sequence repeat; RAPD = random amplied polymorphic DNA.
Table 5. Primer sequences, number of polymorphic bands, percent of polymorphism, and polymorphic
information content (PIC) values obtained with SSR and RAPD markers.

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M.D. Shah et al.
Phylogenetic relationship among Botryodiplodia theobromae isolates
The combined data set of DNA markers obtained from all the isolates with SSR and
RAPD markers were analyzed with UPGMA by the NTSYS 2.0 software and a dendrogram
was constructed (Figure 2). All the isolates were divided into three major groups. Group I con-
sisted of 6 isolates (B-PL1, B-PL2, B-PA1, B-PA2, B-PH1, and B-PH2) with 65% genetic simi-
larity. Group II, comprised of 5 isolates (B-PL3, B-PL6, B-PA3, B-PL4, and B-PL5), exhibited
genetic similarity of 73-80%, whereas group III, comprised of 2 isolates (B-ML and B-MH),
had genetic similarity of 79.5%. At 76% of genetic similarity coefcient, Group I was divided
into 3 subgroups with 2 isolates in each subgroup, i.e., Ia (B-PL1, B-PL2), Ib (B-PA1, B-PA2) and
Ic (B-PH1, B-PH2), while Group II was divided into subgroup IIa with 3 isolates (B-PL3, B-PL6,
B-PA3) and isolates B-PL4 and B-PL5 produced individual lineages at 76% similarity coefcient.
Figure 2. Dendrogram obtained from the combined data set of 13 Botryodiplodia theobromae isolates using SSR
and RAPD markers.
DISCUSSION
Die-back and bark canker, caused by B. theobromae, is one of the important diseases
of pear, which has become a major constraint to pear production during the last few decades
(Verma and Cheema, 1984). The variability in disease symptoms, host range and geographical
location of B. theobromae isolates suggests that there might be several strains of this species.
The identication of B. theobromae isolates has proven to be tedious due to absence of stable
morphological and physiological characteristics, which are inuenced by the environment.

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Morphological, pathological and molecular variability in B. theobromae
The differentiation of fungal isolates has been eased by the use of molecular markers as being
stable and highly polymorphic (Barnes et al., 2001; Zhou et al., 2002; Burgess et al., 2003;
Slippers et al., 2004).
The morphological and pathological characterizations of 13 isolates of B. theobromae
collected from different pear growing regions of the Punjab State revealed that these isolates
were fast to moderately fast growing, produced raised to uffy colonies and dark black pyc-
nidia arranged either on the periphery, centered or scattered in the colonies. These isolates
showed varied pycnidia and pycnidiopore characters and demonstrated differences in their
pathogenic potential. Some other authors have also reported morphological and pathologi-
cal variability among isolates of B. theobromae from different hosts (Luke and Paul, 1982;
Sabalpara et al., 1991; Cedeno et al., 1995; Ko et al., 2004; Mali et al., 2005). Although mor-
phological and pathological variability existed among the isolates studied, the least similar
relationship existed between morphologically similar isolates and their pathological behavior.
Based on molecular characterization of B. theobromae isolates, it is evident that the
isolates prevalent on pear cultivars Patharnakh and Baggugosha in Ludhiana, Amritsar and
Hoshiarpur districts have a high level of similarity and are grouped in one cluster. The iso-
lates collected from cultivars Punjab Beauty, LeConte and Kieffer also showed a high level
of similarity. Isolate from cultivar Smith was different from the isolates collected from other
pear cultivars but showed more similarity with B. theobromae isolates, collected from mango
cultivar Dashehri. The isolates collected from Patharnakh and Baggugosha exhibited a very
high level of variation from the isolates collected either from mango cultivar Dashehri or from
other pear cultivars. It was observed that different isolates of B. theobromae collected from
pear and mango were not location specic, rather they were genotype specic. The results
are more or less similar to those obtained from a morphological and cultural basis. Burgess
et al. (2003) also found relationships among Botryosphaeria rhodina isolates, from pines and
Eucalyptus, to be more closely linked to the host than to the geographical origin by analyzing
the isolates from South America, Mexico and Indonesia. Mohali et al. (2005) observed low
genotypic diversity in Lasiodiplodia theobromae populations from Venezuela and South Af-
rica. However, the genotypic diversity was very high due to very high gene ow between the
populations from different hosts.
RAPD markers were found to be more efcient in detecting polymorphism among
the isolates than SSR. RAPD markers showed the best polymorphic DNA banding pattern.
We observed that B. theobromae isolates collected from pear growing regions of the Punjab
State are quite variable at the genetic level and variety specic grouping could be achieved to
a greater extent.
ACKNOWLEDGMENTS
The authors would like to thank the Head of the Department of Plant Pathology and
the Head of the Department of Plant Breeding, Genetics and Biotechnology, at Punjab Agri-
cultural University for providing the facilities to carry out the present study.
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