Pathogenic Variability within Indian Alternaria brassicae ...

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: [email protected]

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

http://orcid.org/0000-0002-1875-3639

http://orcid.org/0000-0002-1875-3639

http://orcid.org/0000-0002-1875-3639

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


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]



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.



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

–



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



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.



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.



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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