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ThesisItem Open Access Identification of noval donors for resistance against Tilletia indica causing Karnal bunt from wild and related species of wheat(Punjab Agricultural University, Ludhiana, 2020) George, Anju Grace; Vineet KumarKarnal bunt (KB) of wheat caused by the fungus Tilletia indica is an internationally quarantine-significant disease of wheat. KB can be managed in a better way by exploiting genetic resistance in wild and related species of wheat. A population of 490 lines derived from progenitor species of wheat such as Aegilops tauschii and Triticum spp. (T. monococcum, T. boeoticum, T. dicoccoides), non-progenitor species Triticum araraticum and Aegilops triuncialis and triticale introgressions with wheat was utilized in this study. This study was conducted at the Experimental farm and laboratories, Department of Plant Breeding & Genetics, PAU Ludhiana during rabi seasons of 2018-19 and 2019-20. Among the progenitor and non-progenitor species and their bread wheat introgressions, Triticum araraticum 4747 /WHD 943 // bread wheat showed the highest level of KB resistance. This was followed by PDW 233 / Aegilops tauschii 14119// bread wheat. Among the triticale lines with diverse genetic background and their introgressions with bread wheat, TL 2908/2*CS(S)// DBW17 showed the highest level of KB resistance followed by T 3021/2* CS (S) // PBW 550. These lines were considered as promising donors for KB resistance and can be utilized for further breeding disease resistant varieties. Expressed Sequence Tag (EST) based markers specific for chromosome arms of rye were used to determine rye chromosomes associated with the KB resistant triticale x bread wheat lines. The introgressed regions of rye chromosome 4RL, 4RS and 7RL might have contributed to KB resistance in the phenotypically resistant lines.ThesisItem Restricted Bio-management of chickpea wilt incited by Fusarium oxysporum f.sp. ciceri (Padwick) Synd. & Hans.(Punjab Agricultural University, Ludhiana, 2020) Arpanpreet Singh; Narinder SinghFusarium wilt of chickpea caused by Fusarium oxysporum f.sp. ciceri is a serious disease in chickpea growing areas of Punjab. Rhizosphere soil samples were collected from chickpea fields of Ludhiana, Gurdaspur, Mansa, Moga and Bathinda regions and analysed for antagonistic microflora. Five isolates of Trichoderma spp. and three isolates of Pseudomonas spp. were isolated and screened in vitro for antagonistic activity against Fusarium oxysporum f.sp. ciceri causing wilt of chickpea. Th5 isolate of Trichoderma viride and Pf2 isolate of Pseudomonas aeruginosa showing maximum inhibition of 73.11 and 67.63 per cent against Fusarium oxysporum f.sp. ciceri in dual culture assay were selected. Th5 isolate also showed highest chitinase activity 8.85 unit/ml and Pf2 showed highest siderophore activity with the production of 29.07 mm diameter of orange to yellow coloured halo. Efficacy of talc based bioformulations of Trichoderma viride and Pseudomonas aeruginosa isolates applied as seed, soil and seed + soil treatment was seen against Fusarium wilt of chickpea under field and pothouse conditions. Maximum disease inhibition was observed in chemical treatment (Carbendazim) with 88.95, 88.12 and 88.72 per cent in Ludhiana, Gurdaspur and Pothouse trial, respectively, followed by the Trichoderma viride (Th5) seed + soil treatment with 81.84, 85.69 and 83.63 per cent disease inhibition in Ludhiana, Gurdaspur and Pothouse trial, respectively. Maximum growth promotion was also recorded in chemical treatment (Carbendazim) i.e. shoot length, root length and ultimately increased the yield of chickpea under both field and pothouse conditions followed by the isolate Trichoderma viride (Th5) seed + soil treatment. The rhizospheric competence of the antagonists showed that enough population density of antagonists was present throughout the crop period to control the pathogen both under both field and pothouse conditions. Maximum shelf life of talc-based powder bioformulation of all the antagonists was recorded in low temperature conditions (0°C) when packaged in polythene bags.ThesisItem Restricted Identification and validation of molecular markers linked to loose smut resistance in near isogenic lines in the background of PBW343(Punjab Agricultural University, Ludhiana-, 2020) Harpreet Singh; Ritu BalaLoose smut (Ustilago segetum tritici), an internally seed borne disease, causes 100 per cent losses in wheat (Triticum aestivum). Development of molecular markers and understanding the genetic aspects of resistance to U. tritici resistant wheat lines are major breakthroughs to utilise the loose smut resistance genes in breeding programmes. Therefore, two NIL populations derived from W 5100 x PBW 343 and W 4461 x PBW 343 were phenotyped against loose smut using standardized go-go method using two U. tritici isolates T11 and T34. The genetic analysis revealed that segregation of resistant and susceptible NILs had goodness of fit to the expected segregation ratio of 1R: 1S for both the crosses against both the races. This clearly indicated that a single dominant gene to be responsible for loose smut resistance in wheat in these two donors i.e. W 4461 and W 5100. The mapping of these populations for loose smut resistance in wheat following Bulk segregant analysis identified two markers Xgwm124 and Xwmc382 in NIL population derived from W 5100×PBW 343. The validation of these two markers on the individual NILs revealed the association of 1B and 2A chromosomes with loose smut resistance in the NIL W 5100×PBW 343. The NILs will be further used for fine mapping using SNP genotyping for the complete dissection of these NILs with respect to loose smut resistance.ThesisItem Open Access Characterization of Tilletia barclayana causing kernel smut of rice and its management(Punjab Agricultural University, Ludhiana, 2020) Babanpreet Singh; Anju BalaKernel smut of rice caused by Tilletia barclayana is a designated seed borne disease under Indian minimum seed certification standards. The pathogen converts the grains into black powdery mass resulting considerable loses in the yield as well as quality of the produce. The short duration rice varieties are more susceptible to kernel smut as compared to the other varieties. The present studies aimed at characterization of the populations of T. barclayana isolates obtained from different varieties under cultivation in Punjab from different agro-climatic regions and to devise management of this disease. A total of thirty three isolates of T. barclayana were obtained from thirteen districts and eight different varieties for morphological and pathological characterization. Based on the morphological and cultural characteristics, the isolates were grouped into 8 clusters, from each cluster representative isolates were selected for their pathological characterization under field conditions. Fourteen isolates were used for pathological characterization using spray inoculation technique at fifty per cent flower opening and by syringe inoculation technique at late boot stage. Multivariate cluster analysis of the pathogenicity data grouped the isolates into five and three clusters by spray and syringe inoculation method respectively. Isolate Tb 24 obtained from variety PR 124 from district Ropar was the most virulent isolate of T. barclayana whereas isolate Tb 31 obtained from variety PR 121 from district Tarantaran was the least virulent among the isolates under study. The germinated teliospores obtained from weed hosts, Dichanthium annulatum, Cyanodon dectylon were inoculated on variety PR 114 by spray inoculation method to study the cross infectivity of T. barclayana from weeds to rice. Neither of the isolate from any weed hosts were able to produce disease symptoms on rice. Six different fungicides including Hexaconazole 75% WG, Propicinazole 13.9% + Difenconazole 13.9% EC, Hexaconazole 4% + Zineb 68%, Tebuconazole 50% + Trifloxystrobin 25% WG, Azoxystrobin 23% SC, Propiconazole 25% were evaluated against T. barclayana under lab conditions and for disease control under the field conditions. Minimum ED 50 (3.57 ppm) and ED 90 (37.11 ppm) values were recorded for Hexaconazole 75% WG. Maximum disease control under field conditions was achieved in plots where two sprays of combination fungicide Hexaconazole 4% + Zineb 68% was given. The results of two sprays of Propiconazole 25% EC and combination of Tebuconazole 50% + Trifloxystrobin 25% WG @ 0.1 % and 0.04% respectively were equally effective for the management of disease under field conditions Fifty advanced breeding lines of rice were screened for disease reaction under natural conditions. Disease incidence and coefficient of infection was categorized into different response of disease reaction on the fifty advanced breeding lines under study at Ludhiana and Kapurthala. At PAU Ludhiana fourty six lines exhibited resistance response and four lines exhibited highly resistance response. At RRS Kapurthala 41 lines exhibited resistance response, one of the line was susceptible and seven lines were moderately susceptible to kernel smut under natural conditions. The lines showing resistance response can further be evaluated under artificial inoculation conditions in order to obtain resistance sources against the pathogen. The most virulent pathogen isolate Tb-24 may be used for artificial screening of lines against T. barclayana.ThesisItem Open Access Biocontrol of fusarial wilt of bottle gourd(Punjab Agricultural University, Ludhiana, 2019) Ruchika, Ruchika; Narinder SinghThe wilt of bottle gourd caused by Fusarium oxysporum is a serious disease in bottle gourd growing areas of Punjab. Rhizosphere soil samples were collected from bottle gourd fields of Ludhiana, Kapurthala and Bathinda districts of Punjab in order to analyze the antagonistic microflora. Five isolates of Trichoderma and three isolates of Pseudomonas were isolated and screened in vitro for their antagonistic activity against Fusarium oxysporum. Among the various isolates, two isolates of Trichoderma (T1 and T2) and one isolate of Pseudomonas (P1) showed good inhibition against Fusarium oxysporum under lab conditions. Trichoderma isolate T2 showed maximum inhibition of Fusarium oxysporum (76.33 % and 65.83%) in dual culture and volatile assay, respectively. On Molecular characterization the selected isolates T1 and T2 were identified as Trichoderma asperellum and isolate P1 was identified as Pseudomonas aeruginosa. The selected isolates were then applied as seed, soil and seed + soil treatments using 10g and 15g formulation of each antagonist to check their efficacy against Fusarium oxysporum in two different seasons (March and June) under field conditions. Maximum germination (91.67% and 87.50%) and maximum disease inhibition (92.31% and 86.67%) was found in case of chemical treatment followed by Trichoderma asperellum isolate T2 with a germination of 87.50 per cent and a disease reduction of 84.62 and 80.00 per cent in March and June, respectively. Maximum yield (235.99q/acre and 229.00q/acre) along with maximum total length (5.43 m and 4.83 m) was obtained in case of Trichoderma asperellum isolate T2 in March and June, respectively due to disease control and the plant growth promoting behaviour of the antagonist. The results of rhizospheric competence showed that enough propagules of the antagonists were present throughout the growing season to effectively manage the wilt pathogen. Talc based bioformulation of the selected antagonists showed maximum shelf life on storing at 0°C and packing in polythene bags.ThesisItem Open Access POTENTIAL OF NATIVE STRAINS OF BIOAGENTS FOR THE MANAGEMENT OF DAMPING OFF IN ONION AND TOMATO(Punjab Agricultural University, Ludhiana, 2020) Suma, Moka; Narinder SinghDamping off is a destructive disease in onion and tomato caused by Fusarium oxysporum f.sp.cepae and Rhizoctonia solani in nursery growing areas of Punjab. Rhizospheric soil samples were collected from onion and tomato growing nurseries of Sangrur, Ludhiana and Amritsar districts of Punjab and analysed for antagonistic microflora. Seventeen isolates of Trichoderma and fifteen isolates of Pseudomonas were isolated and screened in vitro against the Fusarium oxysporum f.sp.cepae and Rhizoctonia solani causing damping off in onion and tomato respectively through dual culture technique, volatile and non-volatile compounds. Among all tested isolates Trichoderma isolate (T8) has shown maximum inhibition of Fusarium oxysporum f.sp.cepae (71.42%) and Rhizoctonia solani (84.22%) by dual culture. This isolate also showed maximum inhibition to Fusarium oxysporum f.sp. cepae and Rhizoctonia solani by volatile and non volatile metabolites of Trichoderma isolates. Pseudomonas isolate Pf10 has showed maximum mycelial inhibition of Fusarium oxysporum f.sp.cepae (64.37%) and Rhizoctonia solani (65.93%) by dual culture. It also showed mycelial inhibition of Fusarium oxysporum f.sp.cepae and Rhizoctonia solani by volatile and non volatile compounds released from Pseudomonas isolates. Biochemical tests for estimation of cell wall degrading enzymes has shown the ability of the Trichoderma isolate (T8) and Pseudomonas isolate (Pf10) to produce high level of chitinase (17.57 and 14.20 unit/ml respectively) and β-1,3-glucanase (1.42 and 1.52 unit/ml respectively). Pf10 also showed highest siderophore production of 33.67mm. The efficacy of talc based bioformulations of Trichoderma (T8) and Pseudomonas isolate (Pf10) when applied alone as well as in combination under pot and field conditions was checked against damping off of onion and tomato. In onion under field conditions, Pseudomonas (Pf10) and Trichoderma (T8) when applied as seed+soil treatment @15g/kg showed maximum inhibition of disease (89.99 and 88.75 percent respectively). They also acted as plant growth promoters with root length (14.64 and 14.17 cm), shoot length (25.33 and 25.00cm), fresh weight (4.51 and 4.45g), dry weight (1.82 and 1.80g) and finally yield (195.76 and 193.94 q/acre) of the crop. In tomato under field conditions Pf10 and T8 when applied as seed+soil treatment@15g showed maximum inhibition of damping off disease (88.89 and 85.71 per cent) with yield of 221.86 and 221.40 q/acre respectively. Pf10 and T8 (seed+soil treatments@15g) also acted as plant growth promoters with pooled root length (23.67 and 22.50 cm), shoot length (34.49 and 33.79cm), fresh weight (4.53 and 4.49g) and dry weight (2.22 and 2.19g) respectively during 2018 and 2019. Rhisosphere soil sampling from treated onion and tomato nursery up to 45 days showed decrease in antagonistic population up to 15 days and then it started increasing. The selected antagonists showed promising rhizospheric competency throughout the growing season that was enough to control damping off disease of onion and tomato in both pot and field conditions. Molecular identification of the Trichoderma T8 using endochitinase ech42 gene specific primers was done and found that it is closely related to Trichoderma asperellum and Pseudomonas isolate Pf10 using universal primer and sequence analysis showed similarity with Pseudomonas fluorescens. Study of shelf life of the talc based bioformulation revealed that antagonist Trichoderma asperellum (T8) and P. fluorescens (Pf10) can remain potent for 6 months when stored at room temperature while at low temperature storage (4° and 0°C) they can remain potent upto one year. For Mass multiplication sugarcane press mud, rice leaves and wheat bran supported rapid, maximum growth and sporulation of Trichoderma asperellum T8 at 25°CThesisItem Open Access DIVERSITY AND ECO-FRIENDLY MANAGEMENT OF Helminthosporium spp. OCCURRING ON BARLEY AND OTHER GRAMINACEOUS HOSTS(Punjab Agricultural University, Ludhiana, 2020) Amrinder Kaur; Vineet KumaCereal crops all around the world are reported to be attacked by number of pathogenic microbes, among which the diseases incited by Helminthosporium species are economically very important. Among the 50 diseased samples collected during the survey from the various wheat, barley and oat growing areas of Punjab and adjoining states, Bipolaris sorokiniana was present in all the isolates from barley, wheat and Phalaris minor, while Drechslera avenae was found to be associated with the isolates from oats. All the cultures varied significantly with respect to their morphological traits. Total 12 isolates out of 50 were having greyish black growth of the mycelium on the PDA medium while, 34 and 4 isolates were whitish grey and greyish white, respectively. The pattern and margins of growth in most of the isolates were found to be raised and irregular, respectively. Three types of conidial shapes were recorded in all the isolates viz., oblong, elliptical and slightly curved with maximum conidial germination (95.70%) shown by isolate Bsb1 followed by Bsb42 (92.80%) and Bsw23 (91.28%) and it was minimum in isolate Bsb40 (51.20%) followed by Bsp33 (52.40%). All the 50 isolates were pathogenic towards their susceptible check PL426 (barley), HD-2329 (wheat), OL-9 (oats) and Phalaris minor. Minimum incubation period (2days), maximum no. of lesions per leaf (9.29), maximum size of lesions (9.30 mm2) and highest terminal disease severity score (89) were recorded with isolate Bsb1 from barley, followed by Bsw23 from wheat. The cross- infectivity of representative isolates of each host from each group viz., Bsb1 (barley), Bsw23 (wheat), Bsp34 (P.minor) and Da46 (oats) tested on different hosts revealed that all these isolates were found to be cross-infective on different graminaceous hosts except sorghum, rice and different weed hosts. Molecular variability studied among all the 50 isolates using ten different SSR markers revealed that primer BS 065 and BS 027 were found to be highly polymorphic as compared to other primers and thus can be used further for genetic diversity studies. Maximum melanin (µg/g of mycelium) production was recorded in the isolate Bsb1 (2.58 µg/g) followed by isolate Bsp34 (2.52 µg/g) and Bsw23 (2.49 µg/g) at 25°C, whereas, decrease in the trend of melanin production was observed at lower and higher temperature of 15°C and 35°C, respectively as compared to 25°C and 30°C. Among the different barley genotypes screened under field conditions, five genotypes (BL-1309, BL-1313, BL-1532, BL- 1562 and PL891) were found resistant and thirty three genotypes exhibited moderately resistant reaction to foliar blight pathogen. Considering the harmful effects of only use of fungicidal sprays, an eco-friendly approach was followed to manage foliar blight of barley. Maximum per cent growth inhibition (65.48%) under in vitro conditions was recorded with neem @15% followed by tulsi (45.37%) at the same concentration. Propiconazole 25EC @0.1% (standard chemical check) exhibited 100% mycelial growth inhibition as compared to the different botanicals tested and among bio-agents, T. harzianum exhibited 60.82% growth inhibition of the pathogen followed by T. viride as compared to the control. When these promising botanicals and bio-control agent were tested under field conditions for the management of foliar blight of barley, maximum per cent disease control (48.78%) was recorded with neem as compared to control. Among the different SAR chemicals tested under pot house conditions on two barley cultivars, PL 426 and PL807, maximum disease control (50.96%) was observed when the plants were sprayed with salicylic acid @ 0.03%, followed by 41.90% with BABA @ 0.3%. The promising doses of these SAR chemicals when tested under field conditions were found to be effective in controlling the disease upto 40 – 50% in susceptible cultivar PL 426 and upto 45 to 58% in the moderately resistant cultivar PL 807, as compared to the control and an increase was noticed in activity of defense enzyme (PAL) and phenol content of moderately resistant (PL807) and susceptible (PL 426) barley genotypes inoculated with the spore suspension of B. sorokiniana, however the increase was more pronounced in all the treatments in PL 807 as compared to the susceptible check PL 426. Thus, these disease management strategies could be further incorporated in IPM programmes for effective management of foliar blight disease of barley.ThesisItem Open Access Components of partial resistance against Neck Blast (Pyricularia Oryzae) in basmati rice(Punjab Agricultural University, Ludhiana, 2020) Sohal, Manarshroop KaurThe neck blast disease caused by Pyricularia oryzae was highly prevalent throughout the Punjab state with disease incidence ranging from 5.6 to 52.4 per cent on various basmati varieties. Highest disease incidence was observed in Mahal Kalan village of Barnala district. Amongst varieties, Pusa Basmati 1121 had highest value of disease incidence followed by Pusa Basmati 1401, Pusa Basmati 1509 and CSR 30. Significant variability amongst the neck blast isolates of the pathogen was observed with respect to various morphological and cultural characters viz., colony colour, colony texture, colony margin, mean radial growth (mm), spore size and sporulation intensity. The average sporulation intensity of the isolates on Oatmeal agar medium was 7.55×106 conidia /ml which was higher than the average sporulation intensity of isolates on Paspalum grass medium i.e. 6.97×106 conidia /ml. Three isolates namely NB 55, NB 57 and NB 60 were categorized as fast growing isolates with radial growth of >8mm per day. Various architectural features viz., flag leaf angle, flag leaf length, flag leaf width, panicle axis, panicle diameter, degree of exertion of panicle, culm length, 50% flowering and node aging were recorded in order to find correlation with various disease parameters viz., lesion length, panicle blast severity, area under disease progress curve (AUDPC), incubation period, disease incidence (%) and apparent rate of infection (R). Overall, flag leaf angle, panicle axis and node ageing emerged out to be three important components of partial resistance. Forty five basmati rice genotypes were screened under both artificial and natural conditions against the disease. Four genotypes i.e. Tetep, INGR 15001, INGR 15002 and Pusa Basmati 1637 were found to be moderately resistant to most virulent isolate NB 7 with susceptibility index was below 3 and they could be used as donors in breeding for disease resistance.ThesisItem Open Access Potential of native strains of Bacillus spp. against Fusarium wilt of watermelon(Punjab Agricultural University, Ludhiana, 2020) Geetika; Buttar, Daljeet SinghFusarium wilt of watermelon caused by Fusarium oxysporum f.sp. niveum is a serious disease of watermelon growing areas of Punjab. Rhizosphere soil samples were collected from watermelon fields of Ludhiana, Jalandhar and Kapurthala districts and analysed for antagonistic microflora. Fifty isolates of Bacillus were isolated from the soil samples which were collected from rhizosphere soil of watermelon grown different districts of Punjab using serial dilution plating technique on the mannitol egg yolk polymixin medium. Out of 50 isolates, fifteen isolates of Bacillus were identified and purified based on morphological and biochemical character for further studies. The purified fifteen isolates of Bacillus were screened against the Fusarium oxysporum f.sp. niveum using dual-culture technique, volatile, non-volatiles and siderophore production under the laboratory conditions. B1 and B3 isolates of Bacillus spp. showed maximum (2.26 cm) zone of inhibition against Fusarium oxysporum f.sp. niveum under dual culture technique. These isolates showed 76.94 % and 79.20 % inhibition of pathogen in volatiles and non-volatiles production respectively. B1 isolate of Bacillus also showed highest chitinase and β-1,3 glucanase activity of 2.31 and 1.82 unit/ml, respectively. Similarly siderophore production in terms of halo zone (22.69 mm) was maximum in B1 isolate of Bacillus on Chrome-Azurol S media plate. The sequence of DNA showed on NCBI Blast that the nucleotide homology of the isolate B1 showed similarity with Bacillus subtilis strain 8102 (Accession number HQ123475.1). The isolate B3 based on nucleotide homology exhibited its similarity with Bacillus cereus strain LAR2-3 (Accession number KT151933.1). Efficacy of talc based bioformulations of Bacillus subtilis B1 and Bacillus cereus B3 were examined alone @ 5, 10 and 15g per kg of seed, in soil application ( 15 g per kg of soil) and seed + soil application included combination of both treatments (seed+soil). The chemical Bavistin @1.5 g per kg of seed was taken as standard chemical check. B 3 Bacillus cereus isolate applied as seed + soil @ 15g/kg of seed+soil treatment, showed maximum percentage of seed germination (93.33%), minimum percentage of disease incidence (6.67%) and maximum disease inhibition (87.51%) in watermelon plants in pots. Similar results were observed in B 1 Bacillus subtilis applied as seed + soil treatment @15g/kg of seed+soil showed maximum percentage of seed germination (90.00%), minimum percentage of disease incidence (10.00%) and maximum disease inhibition (81.24%) in watermelon plants in pots in kharif 2018 season. Similar trend was observed in kharif 2019 season. The talcum based bioformulations of these two isolates of Bacillus spp. were also further applied as seed treatment, soil treatment and seed + soil treatment and evaluated against wilt pathogen under field conditions. The maximum seed germination (95.83%) was recorded in both treatments of Bacillus subtilis B1 and Bacillus cereus B3 seed + soil @15 g per kg of seed/soil which was at par with the chemical treatment. The minimum wilt disease incidence (8.33%) was observed in the treatment of Bacillus cereus B 3 seed + soil @15 g/kg seed+soil followed by B1 seed + soil @15g/kg seed+soil treatment was 12.50 per cent. In addition to that these isolates of Bacillus spp. also promoted the plant growth parameter like root length, shoot length, their fresh and dry weight and ultimately increased the fruit yield of watermelon under both pots and field conditions. For the rhizosphere competence of the antagonist soil sampling at 15 days interval was done which showed that desirable colony forming unit count was present throughout the crop period in order to manage the pathogen effectively both under pots and field conditions. Maximum shelf life of talc based powder bioformulation of Bacillus subtilis B1 and Bacillus cereus B3 were recorded at low temperature storage (5°C) in polythene bags package. Therefore the finding concluded that Bacillus subtilis B1 and Bacillus cereus B3 significantly decreased the wilt incidence as compared to untreated control both under pot and field condition respectively. Both of these isolates significantly enhanced plant growth parameter and fruit yield. Hence enhanced the production and productivity of watermelon in Punjab condition.