INDIAN COUNCIL OF AGRICULTURAL RESEARCH DIRECTORATE OF RAPESEED-MUSTARD RESEARCH, BHARATPUR, INDIA

INDIAN COUNCIL OF AGRICULTURAL RESEARCH DIRECTORATE OF RAPESEED-MUSTARD RESEARCH, BHARATPUR, INDIA

Pathogenic variability of Sclerotinia sclerotiorum isolates on Brassica differentials Pankaj Sharma ICAR-Directorate of Rapeseed-Mustard Research Bharatpur, India; pksvirus@gmail.com 2

Rapeseed-Mustard Crop in India English name Indian mustard/ Brown mustard Botanical name Brassica juncea (L.) Czern. & Coss. Indian rape/rapeseed Toria Brassica rapa L. ssp. toria (syn. B.campestris L. ssp. toria) Brown sarson/rape seed Yellow sarson/ Colza/Rapeseed Brassica rapa L. ssp. brown sarson (syn. B.campestris L. ssp. brown sarson) Brassica rapa L. ssp. yellow sarson (syn.b. campestris L. ssp. yellow sarson) Black mustard Brassica nigra (L.) Koch Ethopian mustard Brassica carinata Rapeseed/ Rutabaga Brassica napus L. Taramira/rocket salad Eruca sativa

Cultivated Oilseed Brassicas RAPESEED BROWN SARSON MUSTARD TARAMIRA INDIAN MUSTARD KARAN RAI TORIA GOBHI SARSON YELLOW SARSON

Major Insect and Diseases of Rapeseed-Mustard Sclerotinia rot Alternaria blight Painted bug Aphid White rust

Pathogenic variability of Sclerotinia sclerotiorum isolates on Brassica differentials 6

Sclerotinia sclerotiorum Sclerotinia sclerotiorum (Lib.) de Bary is a ubiquitous necrotrophic fungal pathogen. It is capable of infecting about 500 plant species among 75 families (Sharma et al 2015). In India, it has become a serious problem in many parts of the country like Punjab, Haryana, Rajasthan and Bihar. Sclerotinia has the potential to cause up to 39.4% reduction in yield. This disease gained importance particularly in areas where farmers practiced mono-cropping of Indian mustard, which led to complete crop failure with more than 80 per cent disease incidence recorded in some parts of Punjab Haryana and Rajasthan. 7

Symptoms of Sclerotinia rot on stem, leaf and pod Sclerotia formed on stem, in pith & on root

Variability 9

Morphological and cultural Variability 25 different Indian geographical isolates of Sclerotinia sclerotiorum were studied for their morphological and cultural variability. On the basis of morphological data, isolates were grouped into two major clusters I and II. Cluster II was further divided in four groups A, B, C and D.

Mycelial Compatibility Group (MCG) SR-04 SR-10 SR-03 SR-01 SR-08 SR-07 SR-06 SR-15 SR-05 SR-09 SR-02 SR-14 SR-11 SR-18 SR-12 SR-16 SR-21 SR-19 SR-17 SR-23 SR-13 SR-24 SR-25 SR-22 SR-20 Mycelial compatibility is the ability of two strains of filamentous fungi to anastomosis and form one continuous colony.

Protein profile based variability Total 25 bands were observed having relative mobility (Rm) value ranging from 0.14 to 0.72. The similarity indices for different isolates ranged from 0.32 to 1.0 indicating high variability among the different geographical isolates.

Molecular Diversity 45 Primers produced 692 scorable amplicons. Out of these, 385 fractionated fragments were reported polymorphic. All isolates were placed in to four groups and these major clusters were further divided into sub cluster.

Objectives Resistance in oilseed Brassica against the disease is lacking, only partial tolerance to S. sclerotiorum has been reported (Sharma et al. 2012). Keeping in view, the different Brassica differentials were challenged against 25 geographical isolates of S. sclerotiorum to confirm the variation among pathogen population and genetic difference in host species.

Experiments Twenty five geographical isolates of S. sclerotiorum causing SR of Brassica were collected during 2009-12 from 25 locations in 9 states of India and was maintained in vitro. An experiment was conducted to study the pathogenic variability during 2012-13 at ICAR- Directorate of Rapeseed-Mustard Research, Bharatpur, India.

Nine Brassica differentials i.e. 1. B. juncea (cv. Rohini) 2. B. carinata (cv. Kiran) 3. B. rapa var toria (cv. PT 303), 4. B. rapa var yellow sarson (cv. NRCYS 5-2), 5. B. rapa var Brown sarson (cv. KOS 1), 6. B. nigra (cv. BN-1), 7. B. napus (cv. GSC6), 8. Eruca sativa (cv. T-27) and 9. B. alba were used during the study. These were sown in two replications and 65-70 days after sowing, plants were inoculated with stem inoculation technique. 3-weeks after inoculation the observations on stem lesion length and per cent disease incidence were recorded. 16

Pathogenicity of Sclerotinia isolates Isolate B.juncea (Rohini) B.carinata (Kiran) B. rapa var toria (PT 303) B. rapa var y.s. (NRCYS 5-2) B. nigra (BN-1) B.napus (GSC6) Eruca sativa (T-27) B. rapa var b.s. (KOS 1) B. alba % inf L size % inf L size % inf L size % inf L size % inf L size % inf Lsize % inf Lsize % inf Lsize % inf Lsize SR 1 82.5 17.4 29 1.7 86.6 18.6 90.8 22.5 72.2 12.4 70 4.5 91.1 15.7 69.3 11.1 0 0.7 SR 2 85.4 7.1 51.3 1.5 85.9 14.1 85.3 23 47.7 5.8 80.1 2.6 94.8 23.3 76.3 8.3 0 1.2 SR-3 77.5 13.1 23.6 3.8 89.4 13.2 90.8 22.3 49.7 8.7 80 3.7 90.4 25.5 83.3 18.5 0 1.9 SR-4 82.5 25.1 30 1.7 91.3 30.6 75.0 21.9 52.7 2.7 67.5 6.4 83.9 14.9 61.5 3.5 0 1.5 SR-5 60.8 5.0 19.1 1.0 85 22.9 87.5 18.7 41.9 8.8 42.2 2.2 89.9 17.8 68.3 6.3 0 1.3 SR-6 90.0 19.1 36.9 1.4 91.3 29.4 90.8 29.5 45 5.15 43.9 1.3 90.4 21.4 76.3 15.3 0 1.2 SR-7 75.0 13.5 23.6 1.9 90.4 30.7 86.7 17.9 45 1.9 68.7 9 87.3 13.8 64.6 10.2 0 1.1 SR-8 86.6 18.9 22.5 0.9 90.4 23.5 68.5 24 62.7 16.8 32.5 1.5 90.4 18.8 80.4 29.6 0 0.5 SR-9 76.3 12.5 42.2 0.9 56.3 12.6 91.3 26.2 40 4.1 36.9 1.4 86.6 23 51.3 5.0 0 0.9 SR-10 90.0 25.1 59.0 1.5 87.4 23.7 85.0 19.3 52.2 29.8 48.7 2 85.4 20.7 30.6 1.2 0 0.7 SR-11 76.2 5.7 27.5 0.9 67.2 7.3 77.2 28.8 52.7 12.7 55 3.4 90 15.6 66.6 6.2 0 0.8 SR-12 80.9 12.0 33.6 3.3 88.7 26.6 76.3 19.1 52.8 2.8 56.9 5.3 80.9 17.1 49.4 4.9 0 1.0 SR-13 66.8 5.9 27.5 1.4 90 27.5 90.9 24.3 40 2.05 22.5 0.5 83.9 9.8 60 8.4 0 0.3

Isolate B.juncea (Rohini) % inf L size % inf B.carinata (Kiran) L size % inf B. rapa var toria (PT 303) L size B. rapa var y.s. (NRCYS 5-2) % inf L size % inf B. nigra (BN-1) L size B.napus (GSC6) % inf L size Eruca sativa (T-27) % inf L size B. rapa var b.s. (KOS 1) % inf Lsize % inf B. alba L size SR-13 66.8 5.9 27.5 1.4 90 27.5 90.9 24.3 40.0 2.0 22.5 0.5 83.9 9.8 60.0 8.4 0 0.3 SR-14 66.3 8.6 25.0 0.3 85.9 22.9 78.4 26.3 50.0 2.3 71.2 0.7 81.3 15.3 65.0 3.8 0 0.5 SR-15 80 4.4 28.6 0.8 78 8.9 73.3 4.5 55.6 4.2 56.2 0.7 80.0 11 80.0 14.0 0 1.2 SR-16 90.0 17.9 33.6 2.5 90 22.8 77.0 19.4 59.1 3.0 66.3 7.8 78.0 12.1 65.0 5.9 0 0.7 SR-17 77.5 11.6 27.5 2.8 85 18.0 86.3 15.0 83.3 13.7 59.0 2.4 80.0 16.2 65.0 11.3 0 1.4 SR-18 63.3 5.5 20.0 1.8 91.3 31.8 85.4 15.9 38.2 2.7 59.0 3.2 85.9 19.2 80.9 15.4 0 1.5 SR-19 60.0 4.1 18.2 2.7 77.8 10.7 80.0 19.1 41.7 6.8 53.8 4.7 81.8 18.1 58.3 6.8 0 1.3 SR-20 71.3 3.6 22.0 1.7 90.4 8.1 91.1 26.8 55.0 11.6 80.0 11.5 91.6 9.2 68.9 5.9 0 1.3 SR-21 76.1 9.3 24.8 2.7 76.3 10.5 91.6 31.5 55.0 2.2 47.2 3.3 91.3 20.5 91.6 16.2 0 0.7 SR-22 60.0 12.5 21.1 0.4 64.7 5.5 65.0 10.6 45.4 4.4 72.5 13 91.9 25.6 59.0 6.3 0 1.1 SR-23 65.1 9.0 21.1 1.0 85.9 22.0 90.4 34.6 38.2 2.4 47.7 1.4 91.6 19.8 47.7 1.4 0 1.0 SR-24 78.4 17.2 19.1 1.0 90.9 39.0 78.8 17.4 57.2 5.9 57.8 1.4 91.3 12.9 70.8 4.2 0 0.8 SR-25 85.0 18.8 23.6 1.2 75.0 13.7 80.4 36.7 50.0 16.3 40.9 1.7 85.0 16.0 55.0 5.3 0 0.7 19

% Sclerotinia infection on different Brassica 20

Lesion size on different Brassica

Variability in different Sclerotinia geographical isolates Minimum dissimilarity =17.35108 (between SR4 and SR16) Max Dissimilarity = 66.66762 (SR-10 and others) 23

Factorial analysis of geographical isolates 24

All the 25 different geographical isolates showed significant variation in stem lesion length (cm) and per cent disease incidence. Based on pathogenic variability the isolates can be grouped as: highly virulent (SR-06 and SR-10), virulent (SR-01, SR-02, SR-04, SR-08, SR- 12, SR-15 and SR-25), moderately virulent (SR-03, SR-07, SR-09, SR-11, SR-17, SR-20, SR-21 and SR-24) and less virulent (SR-05, SR-13, SR-14, SR-18, SR-19, SR-22 and SR-23). 25

Highly susceptible Brassica differential were all var of B. rapa and E. sativa while highly tolerant was B. alba (lesion size 0.5-1.9 cm). 26

Morphological variability and genetic diversity of different geographical isolates of S. sclerotiorum were already proved. The present study demonstrated existence of pathogenic variability among the geographical isolates which could be helpful to design resistance breeding for S. sclerotiorum in oilseed Brassica. 27

My sincere Thanks to: Indian Council of Agricultural Research (ICAR), New Delhi Department of Science and Technology (DST), Govt. of India Organizers 14 th International Rapeseed Congress Director, ICAR-Directorate of Rapeseed-Mustard Research, Bharatpur All scientists and technical staff of DRMR 28