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ThesisItem Open Access RESPONSE OF COTTON GENOTYPES TO RELATION TO INDUCTION OF RESISTANCE AGAINST COTTON LEAF CURL DISEASE(PAU Ludhiana, 2013) Ritu Raj; P S, SekhonThe present study was conducted to check the response of different American cotton cultivars namely RS 921, LH 2076, PIL 8, Ankur 3028 BGII and a desi cotton variety LD 694 to Jasmonic acid (JA) and Salicylic acid (SA) in induction of resistance against cotton leaf curl disease (CLCuD). Different cotton cultivars were grown in pots under screen cages. At four to six leaf stage potted plants of different cotton cultivars were treated with different concentration of JA and SA i.e. 50 μM, 100 μM, 150 μM, 200 μM respectively and water sprayed plants of corresponding genotypes served as control. Quantification of proteins was done using spectrophotometric method from the leaf samples collected at 24, 48, 72, 96 hrs and a week after treatment with JA and SA. Application of JA and SA resulted in the induction of proteins 24 hrs after treatment. After a week interval at 150 μM concentration of JA maximum protein induction of 19.9 mg/g fr. wt., 16.7 mg/g fr. wt., 19.6 mg/g fr. wt., 15.0 mg/g fr. wt. and 14.4 mg/g fr. wt. was recorded in RS 921, LH 2076, PIL 8, Ankur 3028 BGII and LD 694 respectively whereas, in case of SA 200 μM concentration showed maximum protein induction i.e 10.6 mg/g fr. wt., 9.3 mg/g fr. wt., 9.9 mg/g fr. wt., 9.0 mg/g fr. wt., 8.1 mg/g fr. wt. in RS 921, LH 2076, PIL 8, Ankur 3028 BGII and LD 694 respectively. In the induction of proteins JA was found to be more effective than SA. Electrophoretic study of cotton cultivars treated with 150 μM of JA and 200 μM of SA revealed the induction of Pathogenesis-related (PR) proteins ranging from 15-45 kDa along with some other proteins as well. JA and SA applied at different concentrations affected the CLCuD incidence and severity when inoculated with viruliferous whiteflies (Bemesia tabaci) in screen cages. Lower disease incidence as well as disease index was observed with JA when applied @ 150 μM and SA @ 200 μM. Disease incidence was 37%, 30%, 30% and disease index was 48%, 40%, 40% in RS 921, LH 2076, Ankur 3028 BGII at 150 μM concentration of JA whereas, at 200 μM SA disease incidence was 48%, 36%, 34% and disease index was 57%, 50%, 50% respectively in above mentioned cultivars and in their respective control values for disease incidence and disease severity were quite high. So, JA at 150 μM and SA at 200 μM concentration was found to be most effective in lowering the disease. Latent carry over detection of symptomless plants treated with 150 μM of JA and 200 μM of SA through PCR amplification using DNA specific primers confirmed the presence of virus in all the tested cotton cultivars except LD 694 which signified that PR proteins does not eliminate virus. JA and SA application resulted in imparting tolerance with the induction of PR proteins but does not lead to complete resistance against the disease.ThesisItem Open Access RESPONSE OF COTTON GENOTYPES TO JASMONIC ACID IN RELATION TO INDUCTION OF RESISTANCE AGAINST COTTON LEAF CURL DISEASE(PAU Ludhiana, 2013) Raj, RituThe present study was conducted to check the response of different American cotton cultivars namely RS 921, LH 2076, PIL 8, Ankur 3028 BGII and a desi cotton variety LD 694 to Jasmonic acid (JA) and Salicylic acid (SA) in induction of resistance against cotton leaf curl disease (CLCuD). Different cotton cultivars were grown in pots under screen cages. At four to six leaf stage potted plants of different cotton cultivars were treated with different concentration of JA and SA i.e. 50 μM, 100 μM, 150 μM, 200 μM respectively and water sprayed plants of corresponding genotypes served as control. Quantification of proteins was done using spectrophotometric method from the leaf samples collected at 24, 48, 72, 96 hrs and a week after treatment with JA and SA. Application of JA and SA resulted in the induction of proteins 24 hrs after treatment. After a week interval at 150 μM concentration of JA maximum protein induction of 19.9 mg/g fr. wt., 16.7 mg/g fr. wt., 19.6 mg/g fr. wt., 15.0 mg/g fr. wt. and 14.4 mg/g fr. wt. was recorded in RS 921, LH 2076, PIL 8, Ankur 3028 BGII and LD 694 respectively whereas, in case of SA 200 μM concentration showed maximum protein induction i.e 10.6 mg/g fr. wt., 9.3 mg/g fr. wt., 9.9 mg/g fr. wt., 9.0 mg/g fr. wt., 8.1 mg/g fr. wt. in RS 921, LH 2076, PIL 8, Ankur 3028 BGII and LD 694 respectively. In the induction of proteins JA was found to be more effective than SA. Electrophoretic study of cotton cultivars treated with 150 μM of JA and 200 μM of SA revealed the induction of Pathogenesis-related (PR) proteins ranging from 15-45 kDa along with some other proteins as well. JA and SA applied at different concentrations affected the CLCuD incidence and severity when inoculated with viruliferous whiteflies (Bemesia tabaci) in screen cages. Lower disease incidence as well as disease index was observed with JA when applied @ 150 μM and SA @ 200 μM. Disease incidence was 37%, 30%, 30% and disease index was 48%, 40%, 40% in RS 921, LH 2076, Ankur 3028 BGII at 150 μM concentration of JA whereas, at 200 μM SA disease incidence was 48%, 36%, 34% and disease index was 57%, 50%, 50% respectively in above mentioned cultivars and in their respective control values for disease incidence and disease severity were quite high. So, JA at 150 μM and SA at 200 μM concentration was found to be most effective in lowering the disease. Latent carry over detection of symptomless plants treated with 150 μM of JA and 200 μM of SA through PCR amplification using DNA specific primers confirmed the presence of virus in all the tested cotton cultivars except LD 694 which signified that PR proteins does not eliminate virus. JA and SA application resulted in imparting tolerance with the induction of PR proteins but does not lead to complete resistance against the disease. Key words: Cotton, Cotton leaf curl disease (CLCuD), Jasmonic acid (JA), Salicylic acidThesisItem Open Access VARIABILITY IN Albugo candida (Pers.) Kuntze CAUSING WHITE RUST OF RAPESEED-MUSTARD(PAU Ludhiana, 2011) Yash Pal; P. S., SandhuABSTRACT White rust caused by Albugo candida (Pers.) Kuntze is a serious threat to the production of oilseed Brassica crops in India and around the world. Variation occurs w.r.t. infection of the pathogen on different species of oilseed Brassicas which encouraged us to carry out the studies on “Variability in A. candida (Pers.) Kuntze causing white rust of rapeseed-mustard”. Survey was conducted in different locations (Bathinda, Mansa, Barnala, Faridkot, Muktsar, Sangrur, Ferozepur, and Ludhiana) of Punjab to estimate disease incidence and severity of the disease. The disease incidence and severity of white rust ranged from 44.39-59.36 and 21.92-27.83 per cent, respectively, in different locations of Punjab. A total of 52 isolates were collected from different locations and varieties. These isolates were classified into three groups (AC-I, AC-II and AC-III) on the basis of pustule size, pustule shape and germination of sporangia. Three representative isolates of each group were tested on twelve differential host cultivars (B. juncea, B. nigra, B. napus, B. carinata, B. oleracea, B. tournifortii, B. rapa var. Toria, B. rapa (Brown sarson), B. rapa (Yellow sarson), Raphanus sativus, Sinapis alba and Eruca sativa) of rapeseed and mustard. AC-III group has most virulent and AC-II group showed least virulence based on disease reaction, incubation period, latent period, pustule size, shape, number of pustules per leaf and number of sporangia per pustule. Further, within AC-III group, Ac6 isolate was observed as more virulent than other isolates of same group. Hence, it is concluded that morphological and pathological variation exists in A. candida population from Punjab.ThesisItem Open Access MANAGEMENT OF BOTRYTIS BLIGHT (Botrytis gladiolorum Timm.) IN GLADIOLUS THROUGH CULTURAL AND CHEMICAL MEANS(PAU Ludhiana, 2011) SATYA PRAKASH PANDEY; Premjit SinghThe incidence and severity of Botrytis blight of gladiolus (Botrytis gladiolorum Timm.) and yield of the crop were the minimum in the early- and late-planted crop, as compared to the recommended planting date, i.e. 15th October. Closer spacing resulted in higher incidence and severity of the disease but the corm yield was recorded to be higher in the closer spacing as compared to the wider spacing. Plant spacing of 20x 20 cm was found to be suitable. Incidence and severity of the disease were also found to be high in the un-weeded control. Pre-emergence application of weedicide Stomp at 650 ml/ac, alone, and in combination with one manual weeding at six-leaf stage of the crop, recorded low incidence and severity of the disease and also registered high corm and cormel yields. Two manual weedings, at three- and six-leaf stages of the crop, respectively, also gave good results. Among the non-systemic fungicides, Dithane M-45 showed the best result under in vitro conditions as it completely inhibited spore germination of the pathogen at 100μg/ml, while Kavach and Antracol completely inhibited spore germination at higher concentrations of 500μg/ml. Among the systemic-fungicides, Stroby was the most effective fungicide, followed by Flint and Bavistin. Germ tube length of the fungus was found to be the minimum in Kavach and Dithane M-45. The fungicides Stroby and Flint were found to be the most effective fungicides against Botrytis blight of gladiolus under field conditions. The fungicides Kavach and Dithane-M-45 also showed very promising efficacy.ThesisItem Open Access Population dynamics and pathogenic behaviour of Rhizoctonia solani Kühn in response to rice based cropping system of Punjab(PAU, 2015) Kipsumbai, Pixley Kiptui; Sekhon, P.S.Present investigations were undertaken to study the R. solani population dynamics and pathogenic behaviour under different crop rotations, AG groups and the extent of genetic differentiation and host specialization between infecting populations in different hosts. By using species specific primers the occurrence of Rhizoctonia species was analysed and were identified as R. solani (87.8 %), 11.1% were R. oryzae-sativae and 1.1 % R. oryzae admixed infection with R. oryzae-sativae. Rhizoctonia solani was characterized morphologically as well as at molecular level, which showed presence of high degree of variation among these R. solani isolates. The R. solani isolates were further grouped into anastomosis groups (AGs) by AG subgroups specific primers and it was established that most potato isolates were AG3 and AG2-1. Rice and maize isolates were mostly grouped into subgroup AG1-1A. R. solani rice isolates were studied for their aggressiveness on six rice genotypes and the aggressiveness response of these isolates were further analysed by Mahalanobis D2 analysis and two major groups were observed. Out of all the R. solani isolates, nearly 20% were found to be highly aggressive. Cross infectivity studies revealed that R. solani isolates were cross pathogenic to other hosts except potato and cotton isolates which were not able to cause any disease symptoms on rice. But R. solani isolates were found to be more virulent on their host of origin than any other host under test except in chilli isolate (Cl-40) was found to be highly adaptive to all hosts. Three cropping patterns and 17 R. solani isolates were tested to study the pathogenic behaviour and population dynamics of R. solani isolates under fixed sick plots. A significant interaction was observed between the R. solani isolates and the crops grown in these three adopted cropping systems. The least population build–up after two years of rotation was recorded in the potato-spring maize-Basmati rice cropping system. The overall mean of rice isolates in this system was 44.7 CFU/g of soil, while it was high in the cropping pattern comprising potato –moong bean-rice and wheat ie. 68.1 CFU/g of soil. To find out development of resistance, eighty five out of total 99 isolates under test showed sensitive reaction to propiconazole below 50 ppm concentration in vitro. Only ten isolates showed growth inhibition at 100 ppm and four R. solani isolates were least responsive and showed growth inhibition at 250 ppm concentration. All the isolates were found to be sensitive in their reaction to pencycuron when compared with Tilt 25EC. The ED90 values for most of the insensitive R. solani isolates were found to be in the range of 14 to 26 ppm for pencycuron and 10 to 78 for propiconazole. These preliminary studies indicated that there is development of fungicide resistance in R. solani rice sheath blight isolates as only partial disease control at 0.1% spray concentration of Tilt 25EC was recorded. As no fungicide is recommended for seed treatment in wheat so the two fungicides namely propiconazole and pencycuron were tried and found effective in controlling seed rot and seedling mortality in R. solani sick pots. At the molecular level using interspecific sequence repeat primers, a total number of 79 R. solani isolates were analysed and were divided into three major groups (I, II, III). There was partial relationship observed between the level of aggressiveness of the isolates on rice and phylogenetic groups generated by the ISSR markers.ThesisItem Restricted Management of Sheath Blight of Rice Using Native Strains of Biocontrol Agents and Cloning of Antifungal Gene(PAU, 2015) Heflish, Ahmed Ibrahim Abdelbary Ibrahim; Singh, NarinderSheath blight of rice caused by Rhizoctonia spp. is one of the most important rice diseases worldwide including India. Thirty isolates of Rhizoctonia were isolated from infected rice plants. Genetic diversity of the pathogen was determined by using 11 simple sequence repeats (SSR) molecular markers. The isolates were identified with specific primers at species level. Twenty nine isolates were identified as Rhizoctonia solani while one isolate was found to be as Rhizoctonia oryzae. Native biocontrol agents were isolated from rice rhizospheric soils. Twenty seven isolates of Trichoderma and seventeen isolates of Pseudomonas fluorescens were isolated and screened in vitro against R. solani and R. oryzae causing sheath blight of rice through dual culture technique, effect of volatile and non volatile compounds. Among all tested isolates under in vitro conditions Trichoderma T19 showed the maximum inhibition for R. solani and R. oryzae (65.80 and 74.44 % respectively). Volatile metabolites from Trichoderma T19 also caused maximum inhibition of R. solani (56.91 %) and R. oryzae (91.23%). In case of non-volatile compounds inhibition of the pathogens increased with the increase of culture filtrate concentration from 10 to 50 per cent and T19 with 50 per cent concentration showed the highest percentage of inhibition against R. solani (95.80 %) and R. oryzae (90.74%). P. fluorescens (Pf14) reduced the mycelial growth of R. solani by 60.49 per cent (inhibition zone 9.67 mm) and of R. oryzae by 74.94 per cent with 12.78 mm inhibition zone. Volatile compounds from P. fluorescens (Pf14) showed inhibition of mycelial growth of R. solani and R. oryzae (76.79 and 66.79 % respectively), while in case of non volatile the inhibition by Pf14 using 50 per cent concentration of culture filtrate against R. solani and R. oryzae was 82.47 and 93.46 per cent respectively. Biochemical tests for estimation of cell wall degrading enzymes showed the ability of T19 and Pf14 to produce high level of chitinase (22.1 and 17.5 unit/ml respectively) and β-1,3-glucanase (1.92 and 1.76 unit/ml respectively). Pf14 also presented high activity of siderophore production (20.5 mm halo zone). Fingerprinting of the most effective seven isolates of Trichoderma and of P. fluorescens using SSR marker showed variation between the isolates at molecular level. Efficacy of talc based bioformulations of Trichoderma T19 and P. fluorescens Pf14 applied individually as well as in combination under greenhouse and field conditions was seen against sheath blight of rice. Trichoderma T19 when applied as seed + soil + foliar spray showed the maximum reduction of disease incidence (67.49 %) and disease severity (82.92 %). It also acted as plant growth promoter and increased the number of tillers/hill (12.20), plant height (72.71 cm) and finally the yield of the crop (71.3 q/ha). Molecular identification of Trichoderma T19 using ITS1 and ITS4 universal primers showed 100 per cent similarity with T. asperellum. Cloning of endochitinase42 gene from T. asperellum T19 (potent strain) and Trichoderma T5 (mild strain) showed no difference in the gene sequence between the two Trichoderma isolates, while the difference in the antagonistic activity may be was due to the difference in the promoter region of the gene. Study of shelf life of bioformulations revealed that antagonists T. asperellum T19 and P. fluorescens Pf14 can remain potent for 6 months when stored at room temperature, while at low temperature storage (4oC) these can remain potent up to one year. For mass multiplication sugarcane pressmud and rice leaves supported rapid, maximum growth and sporulation of T. asperellum T19.ThesisItem Open Access Variability in Ustilaginoidea virens (Cke.) Tak. causing false smut of rice and identification of resistance sources(PAU, 2014) Rani, Ritu; Sharma, Vineet KFalse smut of rice (Ustilaginoidea virens) has become an important disease causing significant yield losses in major rice producing states of India including Punjab. Thirty five isolates of U. virens were differentiated on the basis of their morphological, physiological, pathological and molecular characteristics. All the isolates produced well defined colonies on potato sucrose agar (PSA) medium with colony diameter ranging from 26.0 to 90.0 mm after 2 weeks of incubation. Colony color in most of the isolates was initially white which changed to yellow and finally became green. The conidia were spherical, hyaline and warty and size ranged from 4.05-4.94 to 6.10-6.36μm. The isolates also differed in the growth pattern from appressed, fluffy, less fluffy to raised. Sectoring in mycelial growth was observed in five isolates (Uv25, Uv26, Uv27, Uv28 and Uv29). Mycelial growth of representative isolates (Uv1, Uv4, Uv9, Uv13, Uv21, Uv28) of U. virens grown on Czapek‘s dox agar as basal medium was highly variable when supplemented with different carbon and nitrogen sources with maximum utilization of sucrose and sodium nitrate, respectively; consequently resulting in highest growth. Maximum growth was produced when isolates were incubated at 25 ± 2°C (average dry weight = 57.74mg) followed by 30 ± 2°C (56.84mg). Sucrose (2 and 1%) was found superior in inducing spore germination than dextrose. Potato sucrose broth was the best medium for supporting maximum mycelial growth (104.80mg) and sporulation. Maximum colony diameter and sporulation was also observed on PSA (68.74mm) followed by potato dextrose agar (59.26mm). Maximum sporulation was produced on natural substrate medium based on barley seeds (5.54 X 105 conidia/ ml). Among all the isolates, Uv1 was found more aggressive on both the cultivars. Rest of the isolates varied in their virulence/ aggressiveness behavior on tested cultivars and two isolates viz. Uv17, Uv26 and Uv28 failed to produce disease on PR 114. The genetic variation among 35 isolates of U. virens by PCR amplification using ten random operon decamer primers showed polymorphism. Similarity coefficient values among U. virens isolates ranged from 0.56 to 0.97. Of 31 elite germplasm lines three viz. PAU-3965-11-4-1-4-2, PAU-3965-11-4-1-1-2, PR118 were highly resistant while PR122 and PR114 were resistant. Disease incidence, infected panicle and number of balls/plant were increased with increased nitrogen level on rice cultivars viz. PR114 and PR116.ThesisItem Open Access Pathogenic and genetic variability in isolates of Fusarium moniliformeSheld. causing stalk rot of maize (Zea maysL.) and its managemen(PAU, 2012) Harleen Kaur; Chander MohanDisease surveys revealed that Fusarium stalk rot was present in all the maize growing areas of Punjab and disease incidence and severity were comparatively high on private company hybrids (31-Y-45 and Double) as compared to public sector hybrids (PMH 1and PMH 2).Maximum disease incidence and severity were found in the district of Hoshiarpur closely followed by Nawanshahar during both Kharif and spring seasons. Frequency of occurrence of Fusarium moniliformeon stalk rot infected plants was highest followed by Macrophomina phaseolina and Cephalosporium maydis.In all, 56 isolates of F. oniliforme were obtained. They were grouped into seven major clusters based on their cultural and morphological characteristics. Pathological behavior of all isolates of F. moniliforme differed on a set of five inbred lines (LM-13, CM-140, CM-143, CML-25 and CM-600) and four maize hybrids (PMH 1, JH 3459, NK 6240 and 31-Y-45). On the basis of multivariate cluster analysis of pathogenicity data, seven clusters were formed and each cluster represented a specific disease reaction to a particular hybrid/inbred tested. The isolate Fm 10 was found most pathogenic with average disease index (ADI) of 66.6 per cent hereas,the isolate Fm 45 was found least pathogenic with ADI of 42.5 per cent. Cross infectivity studies revealed that the maize isolate of F. moniliforme was pathogenic to wheat and basmati rice and non-pathogenic to peas, non-basmati rice and sugarcane. Likewise, sugarcane isolate of F. moniliforme was found most virulent; wheat and rice isolates moderately virulent and basmati rice and pea isolates were comparatively less virulent on maize. Out of twenty RAPD markers used to study the molecular variability of all 56 isolates of F. moniliforme, 18 showed amplification with a total of 220 amplified fragments. The PIC value was found lowest in primer OPT-9 and highest in primer OPT-12. Dendrogram based on molecular data generated by 18 RAPD primers showed six complete clusters and one independent lineage. When the clusters obtained by morphological, pathological and molecular data werecompared, no association could be obtained among cultural characteristics, virulence pattern and RAPD pattern. Studies of antagonistic potential of native biocontrol agents against F.moniliforme revealed that richoderma harzianumisolate, Th1 showed strong mycoparasitism by completely covering the mycelium of pathogen within 4 days of incubation under in vitroconditions. Two methods of application viz. seed treatment with Th1 @ 20 g/kg seed and amending the soil with FYM supplemented with talc based formulation of T. harzianum found promising in pot house studies, were further tested under field conditions. The maximum disease control and seed germination were obtained when soil was broadcasted with FYM supplemented with T. harzianum@ 2 kg talc/q FYM and amendment of soil with FYM supplemented with T. harzianum@ 100g/hill, respectively. Maximum increase in grain yield was obtained in FYM supplemented with T. arzianum@ 100g/hillThesisItem Restricted EVALUATION OF HOST PLANT RESISTANCE AND CHARACTERIZATION OF VIRUS(ES) CAUSING LEAF CURL DISEASE OF CHILLI (Capsicum annuum L.) IN PUNJAB(2013) Itoo, Asif AhmadChilli is known to be affected by forty five viruses, among them whitefly transmitted begomovirus of family Geminiviridae is considered to be the most destructive virus in terms of yield loss, causing leaf curl disease of chilli. In the present study, survey conducted in major chilli growing areas of Punjab showed that chilli leaf curl disease (ChiLCD) is prevalent in all the four districts surveyed with incidence ranging from 11 to 64%; maximum severity recorded in the months July-August in the districts Patiala and Sangrur. A total of 155 genotypes were screened under natural conditions and the identified resistant stocks were subjected to artificial inoculation. Symptom variability and mixed infections of begomovirus along with beta DNA satellite molecule was found in samples collected from field. Five begomoviruses viz., tomato leaf curl Jodyebpur virus (ToLCJV), tomato leaf curl Palampur (ToLCPV), tomato leaf curl New Delhi (ToLCNDV), tomato leaf curl Karnatka (ToLCKV) and papaya leaf crumple virus (PaLCrV) were found to be associated with ChiLCD in Punjab. Typical begomovirus symptoms were observed in artificially inoculated chilli lines after five weeks of inoculation. Three lines Surian 2010, Perennial and Japanese Loungi were promising against tomato leaf curl Joydebpur virus causing ChiLCD. Symptomatic and asymptomatic weeds showed presence of begomovirus when subjected to rolling circle amplification (RCA) + polymerase chain reaction (PCR), except Calatropis and Parthenium. Weeds are potential reservoir of tomato leaf curl Joydebpur virus (Abutilon and Ageratum), papaya leaf crumple virus (Cannabis), chilli leaf curl India virus (Ipomea and Amranthus) in Punjab.