Post on 29-Jan-2023
ORIGINAL ARTICLE
Pathogenic Variability within Indian Alternaria brassicaeIsolates Using Seed, Cotyledon and Leaf of Brassica SpeciesPrabhu Dayal Meena1 , Riteka Gupta1, Hari Singh Meena1, Pankaj Sharma1 and Sanjay Jambhulkar2
1 ICAR-Directorate of Rapeseed-Mustard Research, Bharatpur, 321 303 Rajasthan, India
2 Nuclear Agriculture & Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400 085 Maharashtra, India
Keywords
Alternaria brassicae, isolates, oilseed
brassica, pathogenic response, variability
Correspondence
P. D. Meena, ICAR-Directorate of Rapeseed-
Mustard Research, Bharatpur, Rajasthan,
India.
Email: pdmeena@gmail.com
Received: June 8, 2016; accepted: January 6,
2017.
doi: 10.1111/jph.12555
Abstract
Thirty Alternaria brassicae (Berk.) Sacc. isolates from diverse geographical
locations of India were studied for pathogenic variability on seed, cotyle-
don and true leaves of Brassica species. Seed germination was reduced
maximum by isolate BAB-39 in Brassica juncea cultivar Varuna (22.1%),
Brassica rapa var. Toria cultivar PT-303 (12%), Brassica carinata cultivar
Kiran (12%), Brassica napus cultivar GSL-1 (11%) and tolerant source of
B. juncea genotype PHR-2 (7%), although least by isolate BAB-49. Maxi-
mum lesion size on leaf was recorded in B. juncea cultivar Rohini (11.2,
16.5 and 16.8 mm) with isolates BAB-09 (Pantnagar), BAB-19 (Bharat-
pur) and BAB-39 (Kangra), respectively, and categorized as highly viru-
lent, while minimum lesion size of 3.2, 3.7 and 3.8 mm was observed
with isolates BAB-47 (Tonk), BAB 49 (Jobner) and BAB 04 (Kamroop),
respectively, considered the weak isolates. On B. alba, BAB-09, BAB-19
and BAB-39 isolates caused maximum lesion size of 3.7, 3.8 and 3.9 mm,
respectively, even though it showed maximum tolerance. In both seed
and cotyledon inoculation method, the per cent Alternaria blight severity
above 80% was observed with isolate of BAB-39 (91.5%), BAB-19
(89.0%), BAB-09 (85.5%) and least in isolate BAB-49 (34.0%). Brassica
seed, cotyledon and leaf showed the similar positive response for catego-
rizing A. brassicae isolates as virulent and avirulent. This information
could be used to the development and assessment of resistant brassica
germplasm, especially with A. brassicae populations exhibiting increased
virulence.
Introduction
Indian mustard [Brassica juncea (L.) Czern & Coss.] is
an important source of edible oilseed in South Asia.
Foliar diseases are the most important limiting factors
for cultivation of oilseed Brassica crops in India. Black
spot or Alternaria blight (AB) caused by Alternaria
brassicae (Berk.) Sacc. is a destructive disease of rape-
seed–mustard causing seed yield losses up to 35 per
cent or higher in India (Meena et al. 2002, 2010).
Alternaria blight is a polycyclic disease, with many
infections occurring throughout the growing season.
It is a foliar pathogen and causes a relatively slow
destruction of host tissues through the reduction of
photosynthetic potential (Saharan et al. 2016). An
infection leads to the development of necrotic lesions,
which sporadically have a target-like appearance due
to growth interruptions caused by adverse conditions.
The fungus resides in the centre of the lesion, which is
surrounded by un-invaded chlorotic halo, a symptom
that is usually observed in the infection process of
necrotrophic pathogens (Thomma 2003). Severity of
the disease does not only depend upon the prevailing
favourable weather conditions, but also depends on
the available inoculum pressure on oversummered
(April to July) infected crop residues in the field
(Saharan et al. 2016). Critical growth stages of the
host for both initiation and development of the
J Phytopathol 165 (2017) 238–248 � 2017 Blackwell Verlag GmbH238
J Phytopathol
pathogen have been studied at 45 and 75 days after
sowing, respectively (Meena et al. 2004). The severity
of AB disease is varying in different regions of the
country either due to genetic variation in the patho-
gen, or favourable weather conditions.
Chemical control is only the option for its manage-
ment because still the complete resistance is lacking in
Brassica species all over the world against this fungus.
Therefore, the resistant cultivars are required to stabi-
lize the productivity and to promote sustainability
without using hazardous chemical control in oilseed
Brassica crops.
The morphological and cultural characteristics
within the isolates of A. brassicae have been reported
by some workers (Mehta et al. 2003; Kaur et al.
2007; Meena et al. 2012). In contrast, pathogenic
variation within A. brassicae population across the
location in India has not been examined in detail. A
systematic examination of pathogenic variation
within A. brassicae populations across growing loca-
tions could lead to a better understanding of
changes in its epidemiology, host–pathogen interac-
tions and pathogen management from year to year.
This information can then be used to develop more
effective fungicides, especially with A. brassicae pop-
ulations exhibiting increased insensitivity to fungi-
cide applications (Sangwan and Mehta 2006; Meena
et al. 2012), and contribute to the development and
assessment of resistant Brassica germplasm (Pratap
et al. 2014).
Various approaches have been utilized to measure
virulence within and among Alternaria species, with
many studies comparing the efficiency of these
approaches. Different inoculation methods were com-
pared to assess virulence by Giri et al. (2013); Meena
et al. (2016) consisting of drop plus agarose, seed
inoculation, cotyledon inoculation, and both seed and
cotyledon inoculation with A. brassicae conidia sus-
pension. Results showed that host inoculated at all
stages was most successful in producing infection.
Similarly, Meena et al. (2012) found true leaf inocu-
lation procedure to be successful in assessing viru-
lence of A. brassicae isolates in India. The objective of
this study was to evaluate pathogenic variability of
naturally occurring A. brassicae populations in AB-
infected oilseed Brassica fields across the country. We
hypothesized that, given pathogen isolate consistency,
the isolates of individuals within populations of
A. brassicae would remain stable between locations.
We expected to find diversity among individual iso-
lates within the population, but expected the major
isolates to remain variable across the locations. Iso-
lates were collected and analysed with pathogenicity
to determine whether any pathogenic variation
existed among the A. brassicae isolates from the same
oilseed brassica host and location within a major bras-
sica production region.
Materials and Methods
Collection of A. brassicae isolates
Black spot infected samples of cultivated oilseed
Brassica spp. were randomly collected from different
geographical locations of India to develop the
national repository of the pathogen. The pathogen
was collected from the elevation of minimum
22°070N to maximum 33°440N latitude (north–south)and lowest 73°040E to highest 93°850E longitude
(west–east). To obtain isolates from the diseased
plant tissue, they were washed 3–4 times in steril-
ized distilled water (DW) and then placed in
1% solution of sodium hypochlorite (NaOCl) for
30–60 s, followed by three times rinsing with dou-
bled sterilized DW. Individual plant tissue plated
aseptically on 90-mm Petri dishes containing potato
dextrose agar (PDA) medium at 25 � 2°C under
alternate 12 h cool white fluorescent light and dark
for 7 days. Thereafter, growing mycelia from margin
of apparently distinct colonies of the leaf spot pieces
on the medium were aseptically transferred into
another Petri dishes containing PDA medium, where
it was grown for 15 days at 25 � 2°C. On the basis
of their conidiophores and conidial morphology as
per (Simmons 2007), the pathogen was identified as
A. brassicae and purified using single spore isolation
method under stereo-microscope (Olympus, Tokyo,
Japan). Alternaria blight disease is one among the
serious problems for the oilseed brassica crops in the
foot hills of Himalayas. The total of 30 representative
isolates of different geographical locations of 12
states of India, viz. Uttar Pradesh, Madhya Pradesh,
Uttarakhand, Rajasthan, Haryana, Jammu and Kash-
mir, West Bengal, Himachal Pradesh, Jharkhand,
Assam, Meghalaya and Nagaland among 219 collec-
tions of A. brassicae cultures (Table 1; Fig 1) were
taken to study the pathogenic response on seed, leaf
and cotyledons of Brassica species. However, some
isolates were collected from the crop cultivated areas
may be from same state due to varying Brassica spe-
cies or varying distance. The total 22 isolates were
submitted to the national repository at ICAR-
National Bureau for Agriculturally Important
Microorganisms, Mau Nath Bhanjan, Uttar Pradesh,
India, with vide accession number NAIMCC-F-02599
to 02620 (Table 1).
J Phytopathol 165 (2017) 238–248 � 2017 Blackwell Verlag GmbH 239
P. D. Meena et al. Pathogenic variability within A. brassicae
Pathogenic response
Effect of culture filtrate of A. brassicae isolates on seed germination
Effect of culture filtrate on seed germination and
growth of plumule and radical were studied at the
ICAR-Directorate of Rapeseed-Mustard Research at
Bharatpur, Rajasthan, India. The culture filtrate of
A. brassicae isolates was collected separately as
described by Otani et al. (1998). The subcultured
dishes were flooded with DW, and the surfaces were
removed with the help of a small paintbrush. The
spore suspension was filtered through three layers of
cheese cloth to eliminate the mycelium debris, and the
spores were washed twice in DW by centrifugation
(Eppendorf 5810R) at 200 rpm for 5 min. For success-
ful infection, the uniform inoculums concentration in
suspension adjusted to 5 9 104 conidia/ml (Doullah
et al. 2006) by conidial count using a haemocytometer
[the total count from 4 sets of 16 corner = (conidia/ml
9104) 9 4 squares from one haemocytometer grid].
Culture filtrate of 30 A. brassicae isolates was taken
to study the variation in pathogenic response and
their effect on seed germination of different Brassica
species (B. juncea cultivar Varuna (susceptible), PHR-2
(Tolerant), B. carinata (Kiran), B. rapa var. Toria (PT
303) and B. napus (GSL-1). One hundred seeds of
each cultivar were soaked in culture filtrates for 24 h.
The treated and untreated seeds were allowed to ger-
minate on moist blotter paper in triplicate under labo-
ratory condition using the method suggested by
Agrawal and Dadlani (1992). The seeds were soaked
only in sterile non-inoculated culture filtrate as check.
After 6 days of incubation at 25 � 2°C, per cent seedgermination as well as seedling vigour was recorded.
Pathogenic response of A. brassicae isolates on true leaves
under field conditions
Reaction of different Brassica genotypes including
B. juncea (Rohini & Varuna, PHR-2), B. napus (GSL-1),
B. carinata (Kiran) and B. rapa var. Toria (PT-303) was
Table 1 Alternaria brassicae isolates infecting Brassica species in India
Isolate code
NBAIM Accession
number
Place and location of
isolate collection
Host
plant
Plant
part
Date of
collection
BAB-02 NBAIMCC-F-02600 Jammu, J&K (32°440 N, 74°540E) B. napus Leaf 15-Feb-05
BAB-03 NBAIMCC-F-02601 Mohanpur, WB (23o60N, 88o300E) B. juncea Leaf 04-Mar-05
BAB-04 NBAIMCC-F-02602 Kamroop, Assam (25°740 N, 93°850E) B. rapa sp.Toria Silique 02-Mar-05
BAB-05 NBAIMCC-F-02603 Mau, UP B. juncea Leaf 15-Mar-05
BAB-06 NBAIMCC-F-02604 Golaghat, Assam (22°070N, 92°060E) B. juncea Leaf 03-Mar-05
BAB-08 – Badh Dhamsya, Jaipur (26°880N, 76°150E) B. juncea Leaf 08-Mar-06
BAB-09 – GBPUAT, Pantnagar (29°030N, 79°310E) B. juncea Leaf 28-Dec-05
BAB-12 NBAIMCC-F-02605 Jagadhary, Haryana (30°170N, 77°300E) B. juncea Leaf 28-Dec-05
BAB-13 – Wajirpur, Haryana (30°60N / 77°170E) B. juncea Leaf 28-Dec-05
BAB-18 NBAIMCC-F-02606 Pantnagar, Uttarakhand (29°030N, 79°310E) B. juncea Leaf 18-Jan-08
BAB-19 – Bharatpur, Rajasthan (27°150N, 77° 300E) B. juncea Seed 22-Jan-09
BAB-20 – Navgaon, Alwar (27°340N, 76°380E) B. juncea Silique 28-Feb-05
BAB-23 – Baharampur, WB (24°060N, 88°190E) B. carinata Leaf 23-Feb-05
BAB-28 NBAIMCC-F-02607 Ri-Bhoi, Meghalaya (25°500N, 90°550E) B. juncea Leaf 02-Mar-05
BAB-29 NBAIMCC-F-02608 Dimapur, Nagaland (25°550N, 93°440E) B. juncea Leaf 05-Mar-05
BAB-39 NBAIMCC-F-02610 Kangra, HP (32°050N, 76°180E) B. carinata Leaf 01-Feb-10
BAB-40 NBAIMCC-F-02611 Kangra, HP (32°050N, 76°180E) B. juncea Leaf 01-Feb-10
BAB-41 NBAIMCC-F-02612 Kangra, HP (32°050N, 76°180E) B. napus Leaf 01-Feb-10
BAB-42 NBAIMCC-F-02613 Jhansi, UP (25°270N, 78°370E) B. juncea Leaf 10-Feb-10
BAB-43 NBAIMCC-F-02614 Hazaribagh, Jharkhand (23°590N, 85°250E) B. juncea Leaf 21-Jan-10
BAB-44 NBAIMCC-F-02615 Bijnor, Uttar Pradesh (29°270N, 78°290E) B. juncea Leaf 30-Jan-10
BAB-45 NBAIMCC-F-02616 Jodhpur, Rajasthan (26°180N, 73°040E) B. juncea Leaf 26-Jan-10
BAB-47 NBAIMCC-F-02617 Tonk, Rajasthan (26°110N, 75°500E) B. juncea Leaf 10-Feb-10
BAB-48 NBAIMCC-F-02618 Kanpur, UP (26°280N, 80°240E) B. juncea Leaf 04-Feb-10
BAB-49 NBAIMCC-F-02619 Jobner, Rajasthan (26°950N,75°340E) B. juncea Leaf 25-Jan-10
BAB-50 NBAIMCC-F-02620 Jhansi, UP (25°270N, 78°370E) B. juncea Leaf 10-Feb-10
BAB-52 – Pantnagar, Uttarakhand (29°030N, 79°310E) B. juncea Leaf 18-Jan-08
BAB-53 – Pantnagar, Uttarakhand (29°030N, 79°310E) B. juncea var rugosa Leaf 18-Jan-08
BAB-54 – Datia, MP (25°670N, 78°470E) B. juncea Leaf 08-Feb-05
BAB-55 – Jhansi, UP (25°270N, 78°370E) B. juncea Leaf 10-Feb-10
BAB-56 – Datia, MP (25°670N, 78°470E) B. juncea Leaf 08-Feb-05
J Phytopathol 165 (2017) 238–248 � 2017 Blackwell Verlag GmbH240
Pathogenic variability within A. brassicae P. D. Meena et al.
tested against A. brassicae isolates under field inocula-
tion conditions. The leaves were wounded to eliminate
the protective effect of the epicuticular wax layer in
order to examine the reaction of the underlying tissues
at 45 days after sowing which has been reported as
critical stage for initiation of the disease (Meena et al.
2004). Three leaves from each genotype were inocu-
lated individually with the help of atomizer with a
conidial suspension of 5 9 104 conidia/ml. Control
leaves were sprayed only with DW. Chattopadhyay
et al. (2005) positively correlated the severity of Alter-
naria blight disease on leaves to a maximum daily tem-
perature of 18–27°C, minimum daily temperature of
8–12°C, daily mean temperature >10°C, >92% morn-
ing relative humidity (r. h.), >40% afternoon r. h. and
mean r. h. of >70% and >9 h of sunshine and >10 h of
leaf wetness in the preceding week. Thus, the favour-
able environmental condition for disease development
Fig. 1 Prevalence of Alternaria blight disease of rapeseed–mustard in India. [Colour figure can be viewed at wileyonlinelibrary.com]
J Phytopathol 165 (2017) 238–248 � 2017 Blackwell Verlag GmbH 241
P. D. Meena et al. Pathogenic variability within A. brassicae
was created by three frequent irrigations. The size of 5
lesions in millimetre was recorded and used for analy-
sis to compare the pathogenic response of each A. bras-
sicae isolate after 7 days of inoculation.
Pathogenic response of A. brassicae isolates on cotyledonary
leaves of B. juncea
To create the absolute inoculum load, the seeds were
soaked of susceptible genotype Varuna of B. juncea to
inoculate with spore suspension of 5 9 104 conidia/
ml concentration at 25 � 2°C overnight with 30 dif-
ferent A. brassicae isolates. After 24 h, 10 seeds were
planted in a row in tiny pores of double-folded moist
germinating paper (Sonar, India) fixed in plastic cell
to hold filter paper (5 cm height) placed in a plastic
tray (30 9 40 9 8 cm size). Trays were incubated at
25 � 2°C under 12-h light/12-h dark cycle with 5.7
Klux intensity for 7 days. Conidial suspension of
5 9 104 conidia/ml was prepared with the help of
haemocytometer by diluting a conidial stock solution
with double sterile DW (Meena et al. 2016). The 6-
day-old seedlings were sprayed with sterilized DW to
clean the surface of cotyledons 24 h prior to inocula-
tion. Cotyledon leaves were inoculated by adding
10 ll drop of 5 9 104 conidia/ml suspension of
A. brassicae isolates with the help of micropipette after
scraping the surface of host by a camel’s hair brush
(Meena et al. 2016). The trays of inoculated cotyle-
dons were then incubated under same conditions for
7 days. The disease reaction prior to inoculation and
7 days after inoculation on individual cotyledon was
recorded as per the scoring to assess the host–patho-gen interaction response (Meena et al. 2016). Accord-
ing to pathogenic symptomatic response of cotyledon
infection, observations were recorded by a rating scale
of 0–7 interaction phenotypic (IP) classes as per Leckie
et al. (1996) with minor modification. Cotyledons
were categorized into eight IP classes, ranges in even
numbers from 0 to 7, that is, 0 = no response,
1 = light small necrotic flecking, 2 = heavy necrotic
flecking, 3 = minute lesions on cotyledon, 4–5 = few
and numerous lesions on cotyledons, and 6–7 = large
scattered or coalescing lesions, including yellowing
and rotting (dead). Per cent AB severity was calcu-
lated using the formula as (Meena et al. 2016):
Per cent disease severity (PDS)
¼ Total sum of numerical ratings� 100
Maximum rating�Number of samples observed
However, the AB disease score was given based on
host response and pathogen growth for different
interaction phenotype.
Statistical analysis
Data from the experiments were subjected to analysis
of variance (ANOVA), and intermean differences
between treatments were established using the least
significant difference (LSD) using statistical analysis
system (SAS) version 9.3 (SAS Institute Inc., Cary, NC,
USA).
Results
Pathogenic response of total 30 A. brassicae isolates
was studied in vitro using different methods at ICAR-
Directorate of Rapeseed-Mustard Research, Bharat-
pur, India. Results indicated that the different isolates
of A. brassicae confirmed varying response in their
pathogenic reaction on different Brassica species indi-
cating the existence of pathogenic variability among
pathogen population prevalent in India. Virulent iso-
lates were identified in the present study for further
use in identification of resistance in oilseed Brassica.
Response of A. brassicae on B. juncea at cotyledonary
leaf stage has been studied first time.
Effect of culture filtrate of A. brassicae isolates on seed
germination
Maximum per cent reduction in seed germination
recorded in B. juncea susceptible cultivar Varuna
(22.1%), B. carinata cultivar Kiran (12%), B. rapa var.
Toria cultivar PT-303 (12%), B. napus cultivar GSL-1
(11%) and B. juncea-tolerant cultivar PHR-2 (7%) by
isolate BAB-39 over control, whereas the maximum
per cent germination was observed in all Brassica spe-
cies with isolate BAB-45 indicating the weak patho-
genic ability of the isolate (Table 2). It was also
observed that the cultural filtrate adversely affected
the radical and plumule growth against A. brassicae
isolates. Significant variability was observed for seed-
ling vigour. Minimum of plumule length (1.3 cm)
was recorded in PHR-2 with isolate BAB-40, while it
was maximum of 8.3, 5.0 and 5.0 cm in B. napus cul-
tivar GSL-1 with isolates BAB-8, BAB-18 and BAB-
23, respectively (Table 3). Radical length was
recorded highest (10.8 cm) in GSL-1 with isolate
BAB-41 followed by 10.7 cm in B. juncea cultivar Var-
una against isolate BAB-18 and lowest with isolate
BAB-43 in all test genotypes (Table 3). Results indi-
cated the existence of variability among different geo-
graphical isolates of A. brassicae with the pathogenic
response of culture filtrate on Brassica species. Simi-
larly, the different Brassica species showed varying tol-
erant response against the different isolates.
J Phytopathol 165 (2017) 238–248 � 2017 Blackwell Verlag GmbH242
Pathogenic variability within A. brassicae P. D. Meena et al.
Table
2Effect
ofseeddeepin
filtrate
ofdifferentAlternariabrassicaeisolatesonseedgerm
inationofdifferentBrassicaspecies
A.brassicae
isolates
%germ
ination
%reduction
overco
ntrol
%germ
ination
%reduction
overco
ntrol
%germ
ination
%reduction
overco
ntrol
%germ
ination
%reduction
overco
ntrol
%germ
ination
%reduction
overco
ntrol
B.junceacv.Varuna
B.junceacv.PHR-2
B.carinata
cv.Kiran
B.napuscv.GSL-1
B.rapassp.Toriacv.PT-303
BAB-02
81.5
5.2
100
089
11
98
298
2
BAB-03
74.0
14.0
97
396
498
296
4
BAB-04
81.0
5.8
100
095
596
4100
0
BAB-06
83.0
3.5
100
095
5100
0100
0
BAB-08
77.0
10.5
96
490
10
100
0100
0
BAB-09
78.0
9.3
100
094
694
695
5
BAB-12
83.0
3.5
95
590
10
96
4100
0
BAB-18
72.0
16.3
100
0100
094
695
5
BAB-19
69.5
19.2
100
090
10
96
4100
0
BAB-20
70.0
18.5
100
094
694
6100
0
BAB-23
79.0
8.1
97
393
790
10
100
0
BAB-28
66.0
11.9
100
093
796
4100
0
BAB-29
76.5
11.0
98
290
10
98
293
7
BAB-39
71.5
22.1
93
788
12
89
11
88
12
BAB-40
80.5
6.4
98
296
496
498
2
BAB-41
70.0
18.6
100
0100
098
2100
0
BAB-42
80.5
6.4
96
488
12
94
6100
0
BAB-43
83.0
3.5
98
291
990
10
99
1
BAB-44
69.0
19.8
97
397
394
6100
0
BAB-45
79.5
7.6
100
0100
096
4100
0
BAB-47
75.0
12.8
97
394
696
4100
0
BAB-48
81.5
5.2
100
0100
094
697
3
BAB-49
83.0
3.5
100
0100
0100
0100
0
BAB-50
79.5
7.6
100
091
998
2100
0
BAB-52
74.0
14.0
98
294
694
6100
0
BAB-54
75.5
12.2
95
593
792
892
8
BAB-55
75.0
12.8
98
290
10
94
696
4
BAB-56
72.5
15.7
100
0100
0100
098
2
Check
86.0
–100
–100
–100
–100
–
LSD(P
=0.01)
4.1
–2.7
–4.2
–4.4
–2.4
–
J Phytopathol 165 (2017) 238–248 � 2017 Blackwell Verlag GmbH 243
P. D. Meena et al. Pathogenic variability within A. brassicae
Response of A. brassicae isolates on B. juncea at
cotyledon leaf stage
In this experiment, per cent AB was recorded with 30
isolates of A. brassicae. Minimum % AB severity was
observed with isolate BAB-49 (21.5%) and maximum
in BAB-19 (73.5%) in 7-day-old seeding with seed
inoculation. However, the infection range reached up
to 34.0–89.0% with cotyledon inoculation by isolates
BAB-49 and BAB-06, respectively, on cultivar Varuna
after 15-day-old seedlings (Table 4). However, the AB
severity recorded after 7 days was 11.5% and after
15 days 13.3% in un-inoculated cotyledons. A. brassi-
cae conidia may germinate on the upper epidermis of
B. juncea cotyledonary leaf by producing germ tubes
which may penetrate the host directly without forma-
tion of an appressorium. The mycelium then ramify
and colonize mesophyll and palisade, tissue, leading
to cell necrosis by producing toxins or metabolites
that form the necrotic lesions. Interaction phenotype
ranged from 0–2 to 0–7 after 7-day cotyledon by seed
inoculation, although it was 0–3 to 0–7 after inocula-
tion of cotyledon. The number of conidia was counted
in cotyledon tissue using microscope (Olympus model
BX51TRF) in the range of 0.0 in un-inoculated to 8.67
conidia per microscopic field at 209 magnification
with BAB-39. Conidia number in host tissues also
proved the existence of variability among the isolates
which coincides with the manifestation of AB disease
on cotyledons.
Pathogenic response of A. brassicae isolates on leaves of
Brassica species under field conditions
Maximum spot size recorded in B. juncea cultivar
Rohini (16.8 and 16.5 mm) showed susceptibility
against BAB-39 and BAB-19 isolates, which seems to
be highly virulent (Fig 2). Maximum tolerance was
observed in B. alba. However, B. alba infected with
the highly virulent isolates BAB-19 and BAB-39
Table 3 Effect of different Alternaria brassicae isolates on plumule and radical length of different Brassica species
A. brassicae
isolates
Plumule length (cm) Radical length (cm)
Varuna PHR-2 Kiran GSL-1 PT-303 Varuna PHR-2 Kiran GSL-1 PT-303
BAB-02 2.7 1.9 2.3 2.8 2.1 8.1 3.1 6.7 6.2 4.0
BAB-03 1.9 2.1 1.9 3.4 2.0 3.0 3.9 3.1 6.3 4.1
BAB-04 4.9 3.4 3.3 4.5 4.0 9.2 8.5 8.0 9.0 7.4
BAB-06 3.1 3.0 2.9 3.4 3.4 10.4 6.5 8.1 7.4 6.9
BAB-08 3.8 3.3 3.4 8.3 3.8 9.5 5.2 8.2 7.1 7.2
BAB-09 2.8 4.3 3.6 3.5 3.8 7.0 3.8 6.0 7.3 6.3
BAB-12 3.7 3.4 3.2 4.4 4.2 7.3 8.0 8.2 7.4 7.0
BAB-18 4.0 3.8 3.4 5.0 4.2 10.7 8.4 8.3 9.3 8.2
BAB-19 4.0 3.4 4.6 4.7 4.0 9.8 8.5 8.0 9.2 7.3
BAB-20 4.5 3.5 3.2 4.4 3.9 10.3 8.6 8.1 7.3 7.0
BAB-23 3.3 3.5 3.3 5.0 3.3 7.5 8.8 7.1 6.1 5.2
BAB-28 4.1 3.5 3.5 4.5 4.2 9.7 8.3 8.7 9.1 8.4
BAB-29 3.5 3.0 3.1 2.7 2.3 9.8 6.7 7.7 8.0 3.9
BAB-39 3.5 3.7 3.1 4.6 4.0 9.2 8.5 8.1 6.9 7.1
BAB-40 2.3 1.3 1.9 2.8 2.3 6.7 2.0 5.8 6.3 5.9
BAB-41 1.8 1.8 3.4 3.9 4.2 3.9 7.7 8.0 10.8 7.4
BAB-42 3.3 3.9 3.0 3.2 3.0 6.5 3.0 6.5 6.3 5.3
BAB-43 2.1 2.2 2.0 1.4 1.8 3.5 2.6 3.4 1.4 1.4
BAB-44 4.3 4.3 3.4 4.3 4.0 8.4 8.2 8.9 8.0 7.3
BAB-45 4.2 3.5 3.7 4.7 4.4 9.3 8.4 8.5 7.3 6.3
BAB-47 3.4 3.0 2.9 3.2 3.0 8.2 7.4 7.9 7.2 6.6
BAB-48 3.8 3.7 4.2 4.6 4.1 9.7 7.9 7.8 10.6 8.2
BAB-49 2.9 2.9 2.5 2.7 3.1 3.9 6.6 7.2 6.9 6.6
BAB-50 4.4 3.3 3.2 4.8 3.9 9.7 7.4 7.9 9.1 6.7
BAB-52 2.7 3.8 2.9 2.9 2.9 5.4 6.0 8.0 6.9 6.0
BAB-54 4.2 3.2 3.4 4.3 1.6 10.0 7.8 8.4 8.7 4.4
BAB-55 2.7 2.7 2.7 3.2 3.9 8.0 8.0 8.0 7.0 7.5
BAB-56 3.6 3.1 3.0 3.4 4.1 7.7 7.8 7.7 7.6 7.3
LSD (P = 0.01) 1.0 0.9 1.3 0.8 1.0 1.7 1.5 1.4 1.6 2.4
J Phytopathol 165 (2017) 238–248 � 2017 Blackwell Verlag GmbH244
Pathogenic variability within A. brassicae P. D. Meena et al.
caused lesion size of 3.8 and 3.9 mm, respectively.
There is positive connection between origin of plant
hosts (Fig 1) where the disease severity which is sev-
ere seems to be by virulent isolates at Bharatpur
(BAB-19) and Kangra (BAB-39).
The least virulent isolate BAB-49 produces small
lesion size in most of the tested hosts. As the origin
plant host was affected with mild severity (Fig 1),
thus the virulence of isolate also seems to be less
pathogenic at Jobner (BAB-49). Based on lesion
size, the different isolates were grouped as: lesion
size 0–4 mm (Group I) = BAB-4, BAB-41, BAB-45,
BAB-47, BAB-49; 5–8 mm (Group II) = BAB-8,
BAB-9, BAB-13, BAB-29, BAB-40, BAB-43, BAB-
50, BAB-54; 9–12 mm (Group III) = BAB-2, BAB-6,
BAB-7, BAB-20, BAB-28, BAB-44, BAB-56; 13–16 mm (Group IV) = BAB-12, BAB-19 and BAB-39
(Table 5). These four groups of isolates of
A. brassicae could be designated as less virulent,
moderately virulent, virulent and highly virulent
based on pathogenic response. However, further
confirmation of variability based on pathogenicity
among isolates is needed using cotyledon, seed and
artificially controlled studies.
Discussion
A detailed understanding of a pathogen’s pathogenic
diversity is imperative to interpreting its contribution
to disease epidemiology and management. Pathogenic
diversity using seed, leaf and true leaf of plant was
observed with 30 isolates of naturally occurring
A. brassicae populations in AB-infected Brassica spp.
fields across the country at the ICAR-Directorate of
Rapeseed-Mustard Research, Bharatpur, India, during
2013–2014 and 2014–2015.
Table 4 Response of Alternaria brassicae isolates on cotyledonary leaf stage of Brassica juncea after seed and cotyledonary inoculation
A. brassicae isolates
Seed inoculation Cotyledon inoculationConidia/microscopic
field at 209IP (n) 0-2,3-5,6-7 % AB severitya IP (n) 0-2,3-5,6-7 % AB severitya
BAB-02 (17, 3, 0) 48.3 (44.1) (13, 7, 0) 54.5 (47.6) 4.00
BAB-03 (17, 3, 0) 66.7 (54.7) (8, 12, 0) 79.0 (62.7) 5.67
BAB-04 (20, 0, 0) 65.0 (53.8) (18, 2, 0) 65.0 (53.8) 4.83
BAB-05 (16, 2, 0) 59.8 (50.8) (12, 6, 0) 64.5 (53.4) 3.83
BAB-06 (9, 9, 0) 60.5 (51.1) (12, 8, 0) 85.5 (67.6) 3.17
BAB-07 (9, 6, 0) 57.5 (49.3) (9, 6, 0) 64.5 (53.4) 5.17
BAB-08 (10, 7, 0) 53.0 (46.7) (13, 4, 0) 67.5 (55.3) 4.83
BAB-09 (13, 5, 0) 68.5 (55.9) (11, 7, 0) 85.5 (67.7) 5.83
BAB-12 (13, 6, 0) 63.5 (52.8) (8, 11, 0) 85.5 (67.6) 7.00
BAB-13 (13, 7, 0) 42.0 (40.4) (8, 12, 0) 65.0 (53.8) 5.33
BAB-19 (17, 3, 0) 73.5 (59.1) (4, 14, 0) 89.0 (70.6) 7.67
BAB-20 (16, 4, 0) 56.0 (48.4) (9, 11, 0) 73.5 (59.1) 5.33
BAB-28 (6, 10, 0) 72.5 (58.5) (5, 12, 0) 83.5 (66.1) 6.83
BAB-29 (12, 6, 0) 59.0 (50.2) (9, 9, 0) 81.5 (64.5) 3.67
BAB-39 (16, 2, 0) 41.0 (39.8) (8, 10, 0) 91.5 (73.7) 8.67
BAB-40 (17, 3, 0) 49.0 (44.4) (11, 9, 0) 73.5 (59.1) 5.67
BAB-41 (19, 0, 0) 63.5 (52.9) (14, 5, 0) 68.0 (55.6) 5.17
BAB-42 (14, 2, 0) 33.0 (35.1) (5, 11, 0) 73.0 (58.7) 4.00
BAB-43 (16, 0, 0) 68.5 (55.9) (13, 7, 0) 81.5 (64.5) 5.33
BAB-44 (16, 4, 0) 43.5 (41.3) (11, 9, 0) 67.5 (55.3) 7.67
BAB-45 (16, 3, 0) 48.5 (44.1) (10, 9, 0) 56.0 (48.4) 3.83
BAB-47 (16, 4, 0) 40.5 (39.5) (11, 9, 0) 68.0 (55.6) 4.83
BAB-49 (18, 2, 0) 21.5 (27.6) (15, 4, 1) 34.0 (35.6) 3.17
BAB-50 (18, 2, 0) 45.0 (42.1) (11, 9, 0) 64.0 (53.1) 4.83
BAB-52 (15, 4, 0) 61.0 (51.4) (3, 16, 1) 66.5 (54.7) 5.33
BAB-53 (16, 4, 0) 48.0 (43.9) (7, 13, 0) 76.5 (61.0) 5.67
BAB-54 (17, 3, 0) 52.0 (46.1) (11, 9, 0) 79.0 (62.7) 5.00
BAB-55 (13, 7, 0) 60.0 (50.8) (0, 20, 0) 81.0 (64.2) 6.83
BAB-56 (8, 9, 3) 47.0 (43.3) (2, 14, 4) 67.0 (56.6) 5.00
Check (18, 2, 0) 11.7 (19.9) (18, 2, 0) 13.3 (21.4) 0.00
LSD(P = 0.01) 4.41 4.79 1.62
aFigures in parentheses are arc sign-transformed values.
J Phytopathol 165 (2017) 238–248 � 2017 Blackwell Verlag GmbH 245
P. D. Meena et al. Pathogenic variability within A. brassicae
Results indicated varying response in pathogenic
reaction on different Brassica species demonstrating
the existence of pathogen variation among A. brassicae
population prevalent in India. Variation among iso-
lates of A. brassicae could be the result of production
of a host-specific toxins which were released during
spore germination on the leaves (Pedras et al. 2009;
Meena et al. 2015), which needs to be further
explored. Virulent isolates including BAB-09, BAB-19
and BAB-39 were identified in the present study for
further use in the detection of resistance in oilseed
Brassica. Response of A. brassicae on B. juncea at
cotyledonary leaf stage has been studied first time. An
important component in host–pathogen interactions
is that characteristics of individuals within a species
are not fixed in their morphology, physiology and
pathogenic response. Pathogen population may differ
in their ability to attack particular host species and in
their physiological adaptations to specific environ-
mental conditions. Although the A. brassicae is an
imperfect fungus, it shows genetic variability which
may be due to the existence of mutation, somatic
hybridization, heterokaryoses, uniform host selection
and extensive dispersal (Saharan et al. 2016).
Fig. 2 Pathogenic response through lesion formation by Alternaria brassicae isolates on true leaf of different Brassica species under inoculated field
conditions. [Colour figure can be viewed at wileyonlinelibrary.com]
Table 5 Classification of Alternaria brassicae isolates based on virulence
Category
Range of %
AB severity A. brassicae isolatesa
Less virulent <60 BAB-02 (Jammu, J&K), BAB-45 (Jodhpur, Rajasthan), BAB-49 (Jobner, Rajasthan)
Moderately virulent 61–70 BAB-04 (Kamroop, Assam), BAB-05 (Mau, UP), BAB-07 (Dausa, Rajasthan), BAB-08 (Jaipur, Rajasthan),
BAB-13 (Wajirpur, Haryana), BAB-41 (Kangra, HP), BAB-44 (Bijnor, UP), BAB-47 (Tonk, Rajasthan),
BAB-50 (Jhansi, UP), BAB-56 (Datia, MP)
Virulent 71–80 BAB-03 (Mohanpur, WB), BAB-09 (Pantnagar), BAB-20 (Navgaon, Rajasthan), BAB-40 (Kangra, HP),
BAB-42 (Jhansi, UP), BAB-52 (Pantnagar, Uttarakhand), BAB-53 (Pantnagar, Uttarakhand), BAB-54 (Datia, MP)
Highly virulent >81 BAB-06 (Golaghat, Assam), BAB-12 (Jagadhary, Haryana), BAB-19 (Bharatpur, Rajasthan),
BAB-28 (Ri-Bhoi, Meghalaya), BAB-29 (Dimapur, Nagaland), BAB-39 (Kangra, HP), BAB-43 (Hazaribagh, Jharkhand),
BAB-55 (Jhansi, UP)
aUP, Uttar Pradesh; MP, Madhya Pradesh; HP, Himachal Pradesh; WB, West Bengal; J&K, Jammu & Kashmir.
J Phytopathol 165 (2017) 238–248 � 2017 Blackwell Verlag GmbH246
Pathogenic variability within A. brassicae P. D. Meena et al.
Although the pathogenic variability in A. brassicae has
been observed by various workers (Saharan et al.
2016), none of these used sufficient pathogen popula-
tion representing the whole country. In the present
study, A. brassicae isolates were collected from differ-
ent geographical sites including 14 states of India
where the oilseed Brassica crops are under cultivation
representing the complete population.
Our results are comparable in the morphological
and cultural variations among A. brassicae isolates
reported by Meena et al. (2012) with their pathogenic
reaction. Culture filtrate medium for selection of
resistance in B. napus against A. brassicicola have been
also used (MacDonald and Ingram 1986). Results indi-
cated the existence of variability among different geo-
graphical isolates of A. brassicae with the pathogenic
response of culture filtrate on Brassica species. Simi-
larly, the different Brassica species showed varying tol-
erant response against the different isolates.
Temperature and r. h. conditions favourable to disease
development noted in the field matched with labora-
tory findings.
Studies on seed as a source of primary inoculums
matched with the reports of Saharan et al. (2016).
Our results showed that the AB infection abundantly
occurs at cotyledonary leaf stage by seed inoculation.
However, Awasthi and Kolte (1989) reported that AB
developed best during rosette to flower stage, which
increased with the age of the host. The production of
host-selective toxins by the necrotrophic fungus
A. brassicae is essential for the pathogenesis (Thomma
2003). Pathogenesis mechanisms are also affected by
a pathogen’s ability to cope with various environmen-
tal stresses, such as reactive oxygen species, pH fluctu-
ation and host defence molecules (Cho 2015).
Conclusion
Pathogenic response on Brassica species found to differ
in their sensitivity against different isolates of A. bras-
sicae. Highest per cent seed germination reduction was
recorded in cultivars Varuna (22.1%), PT-303 (12%),
Kiran (12%), GSL-1 (11%) and PHR-2 (7%) by iso-
late BAB-39 designated as highly virulent over con-
trol (without culture filtrate). Similarly, minimum
plumule length (1.3 cm) was recorded in PHR-2 with
isolate BAB-40, while maximum (5.0 cm) plumule
length was recorded in GSL-1 against isolate BAB-18
and BAB-23. However, radical length was recorded
highest (10.8 cm) in GSL-1 with isolate BAB-41 and
lowest (1.4 cm) in both PT-303 and GSL-1 against
BAB-43 isolate. Maximum lesion size was recorded in
B. juncea cultivar Rohini (16.8, 16.5 and 15.2 mm),
which seems to be more susceptible to BAB-39, BAB-
19 and BAB-09 isolates and seems to be highly viru-
lent pathogen. Maximum tolerance was observed in
B. alba against all isolates. Highly virulent isolates
BAB-09, BAB-19 and BAB-39 were capable to infect
B. alba with lesion size of 3.7, 3.8 and 3.9 mm,
respectively. Similarly, these isolates showed signifi-
cant virulent response at seed, cotyledon and true leaf
stages. Results also indicated that BAB-49 isolate was
found relatively less virulent which produced small
lesion size in most of the test hosts. Based on cotyle-
don AB severity and number of conidia in tissues, the
different isolates showed variability. Results indicated
the existence of variability among different geographi-
cal isolates of A. brassicae with their sensitive effect of
culture filtrate on Brassica species. Highest 8.67 coni-
dia number per microscopic field was produced in
host tissues by highly virulent isolate BAB-39, while
least 3.17 conidia number was produced in host tis-
sues by less virulent BAB-49 isolate. Similarly, the dif-
ferent Brassica species also revealed varying tolerant
response against the different pathogen isolates. There
is positive connection between origins of plant hosts,
where the disease severity which is severe seems to be
by virulent isolates at Pantnagar (BAB-09), Bharatpur
(BAB-19) and Kangra (BAB-39). These locations are
also considered as hot spot for the AB disease in oil-
seed Brassica due to favourable weather conditions.
The least virulent isolate BAB-49 produces small
lesion size in most of the tested hosts. As the origin
plant host was affected with mild severity, thus the
virulence of isolate also seems to be less pathogenic at
Jobner (BAB-49). In the western parts of Rajasthan
and Gujarat, AB disease seems not a problem for this
crop because the weather does not favour the devel-
opment of this pathogen which was also found weak
in virulence.
Acknowledgements
Authors are highly grateful to Indian Council of Agri-
cultural Research (ICAR) for providing financial sup-
port in the form of Institutional project entitled ‘AB
management in rapeseed-mustard’ and Director,
ICAR-Directorate of Rapeseed-Mustard Research,
Bharatpur (Raj), India, for providing laboratory facili-
ties for undertaking the present study.
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