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ThesisItem Open Access Characterization of variability in Cercospora sojina K. Hara causing frogeye leaf spot and identification of resistant sources in soybean(CSK HPKV, Palampur, 2023-01-18) Sharma, Abhilasha; Singh, AmarThe present investigation entitled “Characterization of variability in Cercospora sojina Hara causing frogeye leaf spot and identification of resistant sources in soybean” was undertaken during Kharif season to study pathogen isolation and culture production techniques, development of differential set, pathogen variability (morpho-cultural, pathogenic and molecular), mating type distribution, evaluation of resistant sources, inheritance of resistance, and biochemical basis of resistance against Cercospora sojina. Frogeye leaf spot of soybean (Glycine max (L) Merill) caused by Cercospora sojina Hara emerged as most destructive disease in Himachal Pradesh as PDI recorded was ranged between 0.00 to 95.56 per cent. Thirty isolates of C. sojina were collected from different locations of five districts (Kangra, Mandi, Solan, Hamirpur and Bilaspur) representing 3 agro-climatic zones of Himachal Pradesh. Isolations were done by using three different methods viz; inoculation by bits, inoculation by using moist-chamber technique and inoculation by using toothpick method. Out of these, moist chamber technique was the most reliable as most of the isolates were recovered by using this technique. Thirty isolates were identified as Cercospora sojina based on symptomatology, morphological characters and confirmed with molecular characterization. Culture technique for C. sojina was also standardized as the fungus was slow growing in nature. Among nine tested media, PCDA (Potato Cellulose Dextrose Agar) was found best with 4g concentration of cellulose whereas FRCA (Flat Rice Cellulose Agar) media (4g cellulose) was found best at 25 ± 1ºC to produce long conidia. The pathogen was found variable on the basis of morpho-cultural variability. A differential set was finalized to study the virulence structure of C. sojina. Initially sixty soybean lines were selected from 200 genotypes and tested against six isolates, out of which 25 soybean lines were selected as pre-differential set which was tested against all the 30 isolates of C. sojina and finally a differential set of 10 lines was developed. On the basis of disease reaction on differential set C. sojina isolates were grouped into 10 pathotypes. Virulence analysis of the pathotypes (PCS 1 – PCS 10) revealed that PCS 1 and PCS 2 were highly virulent whereas PCS 3, PCS 8 and PCS 10 were least virulent. Variability studies on morpho-cultural, pathogenic and molecular characters categorized 30 isolates of C. sojina into 11, 10 and 6 groups, respectively. There is no or less correlation among the three types of variability studies. Molecular variability was also studied and analyzed with POPGENE analysis software which revealed that thirty isolates were divided into 2 populations on the basis of geography (Latitude and longitude) and there is substantial gene flow among the two populations. There was occurrence of maximum variability within the population i.e.>90 per cent as PhiPT value ranged between 0.004 - 0.127. Mating type distribution of 30 isolates of C. sojina was studied using MAT1-1 and MAT1-2 primers which revealed the presence of both the idiomorph (MAT1-1 & MAT1-2) found in CSH09 isolate revealed that population in Himachal Pradesh probably undergoing sexual reproduction. Out of a total two hundred lines evaluated under field conditions 30 lines found resistant which were screened against diverse pathotypes (PCS1- PCS-10) and four resistant lines viz.; Harder, JSM 285, CAT 195 and GP465 were identified as highly resistant against all pathotypes. Biochemical basis of resistance was also studied in a resistant line ‘Himso 1685’ and a susceptible line ‘Shivalik’ revealed that the tested enzymes i.e., Phenols, PPO and PO had no or less role in defence as there may be other enzymes which can affect the defence mechanism upon infection in soybean against C. sojina. Inheritance of resistance for C. sojina studied and results revealed that the inheritance was controlled by single dominant gene.ThesisItem Open Access EPIDEMIOLOGY AND MANAGEMENT OF FUSARIUM HEAD BLIGHT OF WHEAT(palampur, 2022-06-30) KAUR, GURPREET; Rana, S. K.The investigation entitled “Epidemiology and management of Fusarium head blight of wheat” was conducted at the Department of Plant Pathology, CSK HPKV, Palampur and Research farm of Rice and Wheat Research Centre (RWRC), Malan during 2019-21. The average disease incidence and severity of FHB at different locations of Himachal Pradesh varied from 3.25 to 7.74 and 15.08 to 25.29 per cent during 2019-20 and 2020-21, respectively. Three Fusarium species viz., Fusarium graminearum, F. compactum and F. avenaceum were found associated with the disease but F. graminearum was more prevalent in all the surveyed locations of Himachal Pradesh. The temperature of 25°C and pH of 5.0 were found to be optimum for mycelial growth, sporulation and perithecia formation of the fungus. The seed and crop residue of maize, wheat and rice served as the primary source of inoculum for the disease development. All the weather parameters viz., temperature, relative humidity and rainfall contributed for the disease development as all of them individually and collectively showed positive correlations with disease severity. Out of 200 tested genotypes of wheat, 13 were found free from disease, 26 as resistant and 54 as moderately resistant to FHB under artificial inoculations. Under in vitro conditions, beejamrit, extracts of Acorus calamus (aqueous and alcoholic), Trichoderma harzianum (TH-5) and carbendazim 50 WP gave highest inhibition of F. graminearum. In a pot experiment, seed treatment and two foliar sprays of carbendazim 50 WP gave maximum reduction (71.85 & 77.16%) in disease incidence and severity, respectively. However, among non-chemical management components beejamrit, aqueous extract of Acorus calamus and Trichoderma harzianum (TH-5) gave maximum reduction in disease incidence and severity. An integrated treatment viz., seed treatment with carbendazim 50 WP (0.2 %) followed by three foliar sprays each of jeevamrit (1 %), aqueous extract of Acorus calamus (1 %) and carbendazim 50 WP (0.1 %) at 10 days interval was found to be most effective giving 57.26 and 61.56 per cent reduction in disease incidence and severity, respectively.ThesisItem Open Access Epidemiology and management of flag smut (Urocystis agropyri (Preuss) Schroet) of wheat(palampur, 2022-05-02) Bhargava, Priya; Rana, S. K.The investigation entitled “Epidemiology and management of flag smut (Urocystis agropyri (Preuss) Schroet.) of wheat” was conducted at the Department of Plant Pathology, CSK HPKV, Palampur and Research Sub Station, Akrot during 2018-21. The average disease incidence at different locations of Himachal Pradesh varied from 2.11 to 17.77 per cent during two consecutive years. Based on molecular characterization, the pathogen was confirmed to be Urocystis agropyri. The maximum spore germination (18.92 %) was recorded after 8 months of inoculum burial in soil while, no germination occurred till 4 months of storage due to the spore dormancy. Inoculation of germinating seed with dry inoculum @ 10 g inoculum/ kg seed proved to be the best method to incite disease with highest incidence of 58.18 per cent. Inoculum load of 20 g inoculum/ kg seed adversely affected the seed health parameters like germination percentage and vigour index of seed as well as growth rate and dry weight of the seedlings with 13.70, 52.96, 54.41 and 6.73 per cent reduction, respectively. However, the maximum reduction in speed of seedling germination/ emergence (15.01 %) was found at 17.5 g inoculum/ kg seed. The maximum incidence of disease (44.87 and 40.64 %) was recorded at soil moisture and temperature of 30 % and 22℃, respectively. Among the weather parameters, the maximum and minimum temperatures were the most important factors for disease development as an increase in the temperature from 11.78 - 22.75℃ at Palampur and 09.20 - 26.13℃ at Akrot increased the disease incidence during both the years. The disease incidence was found to be maximum (13.28 and 16.47%) in 15th November sown crop at Palampur during both the years. While, at Akrot, it was maximum in 15th November sown crop (25.50 %) during 2019-20 and in 30th November sown crop (37.49 %) during 2020-21. Out of 210 wheat germplasm lines/ genotypes, 71 were found immune, 45 as highly resistant and 34 as moderately resistant. The late sowing of wheat was found to be the best with minimum disease incidence of 10.79 per cent followed by early sowing (21.47 %). The sowing depth of 2 cm showed minimum disease incidence of 12.98 per cent. Among the tested organic amendments, Jeevamrit @ 10 ml/ kg soil gave the maximum reduction (82.94 %) in the incidence of flag smut. While, amongst biocontrol agents as seed and soil treatments, resident Trichoderma viride @ 10 g or ml/ kg seed or soil was found to be most effective against the disease with maximum reduction of 47.50 and 34.02 per cent, respectively. From botanicals, the seed treatment with aqueous extracts of Eucalyptus tereticornis @ 15 % concentration was found to be the best with maximum disease control of 79.44, 71.85, 80.30 and 77.55 per cent under two approaches of pathogen inoculation in pot and field experiments, respectively. Among fungicides, tebuconazole 060 FS at 1, 2 and 3 ml/ kg seed, carboxin 37.5% + thiram 37.5% DS, carbendazim 50 WP and carboxin 75 WP at 3 g/ kg seed were found most effective giving complete control of the disease under both the approaches of pathogen inoculation in pot and field experiments. The integration of tebuconazole 060 FS @ 1 ml/ kg seed with Jeevamrit @ 10 ml/ kg soil (as soil treatment); E. tereticornis @ 15 % concentration (as seed treatment); and soil treatment with Jeevamrit and seed treatment with E. tereticornis gave complete control of the disease. On the basis of morphological and molecular characterizations, 17 isolates of U. agropyri were grouped into four groups each i.e. FSG 1 to FSG 4 and FSV1 to FSV4, respectively. The group FSV 1 and FSV 2 were further subgrouped into six subgroups i.e. FSV 1a, FSV 1b, FSV 2a, FSV 2b, FSV 2c and FSV 2dThesisItem Open Access Epidemiology and management of Stemphylium blight of onion(palampur, 2022-04-29) Thakur, Ashima; Banyal, . D.K.The present investigation entitled “Epidemiology and management of Stemphylium blight of onion” was conducted in the Department of Plant Pathology, CSK HPKV, Palampur during 2018-2021with aim to identify disease & pathogen, variability studies, factors affecting pathogen & disease development and evaluation of IDM components viz., chemicals, cultural practices (spacing and date of sowing), plant extracts, biocontrol agents and organic inputs under in vitro, in vivo and field conditions for the management of the Stemphylium blight of onion. During the survey it was observed that disease was prevalent in all the onion growing districts of Himachal Pradesh. The maximum disease severity was observed in district Una (45.4%) followed by Bilaspur (42.5%), Mandi (37.5%) and Kangra (36.9%) whereas, minimum disease severity (35.2%) was observed in district Hamirpur. The disease was identified as Stemphylium blight based on symptoms and pathogen was identified as Stemphylium vesicarium on the basis of morpho-cultural characteristics and molecular characterization. The pathogen variability was studied on the basis of virulence, morphological characters, physiological and molecular characterization, which grouped 32 isolates of S. vesicarium into 6, 14, 12, 12, 9 and 5 groups, respectively. Among different media tested Potato dextrose agar was observed as best media with maximum radial growth whereas, sporulation of S. vesicarium was observed on V8 agar, Oatmeal agar and PDA media. Temperature 250C was found best for the mycelial growth, whereas maximum sporulation occurred at 15-200C. The pathogen perpetuated on debris for quite longer time (8 months) as compared to perpetuation in soil and seeds. The incubation period and latent period of S. vesicarium was observed as 3 & 5 days on detached leaves at 250C and 5 & 7 days on potted plants, respectively. The inoculum level i.e., 3x104 conidia/ml, was found optimum for disease development. Temperature was observed as the most important factor in the disease development having a significant positive correlation with disease severity. Among the eight fungicides viz., trifloxystrobin 25% + tebuconazole 50%WG, azoxystrobin 25EC, difenoconazole 25 EC, carbendazim 50 WP, propiconazole 25 EC, zineb 75 WP, mancozeb 75 WP & copper oxychloride 50 WP evaluated in vitro, propiconazole 25 EC (250 ppm), difenoconazole 25 EC (500 ppm) and trifloxystrobin 25% + tebuconazole 50% WG (1500 ppm) were found most effective which gave complete mycelial inhibition at 250, 500 and 1500 ppm, respectively. Cultural practices revealed that late sown crop at wider spacing resulted into less disease severity with higher bulb yield. In vitro evaluation of plant extracts (aqueous & alcoholic) of Melia azedarach, Lantana camera, Eupatorium adenophorum and Eucalyptus camaldulensis showed that aqueous extract of Eucalyptus gave maximum mycelial inhibition of 53.30 per cent (@ 50%) whereas, alcoholic extracts of all botanicals at 50 per cent concentration except Lantana provided complete mycelial inhibition. In vitro evaluation of bio agents i.e., Trichoderma viride, T. harzianum (JMA-4 and SMA-5), T. koningii (DMA-8 and JMA-11)) and Pseudomonas fluoroscens gave mycelial inhibition ranging from 10.40 to 35.65 per cent. The organic inputs viz; Tamarlassi, Bijamrit, Biosol, Vermiwash, Jeevamrit and Eucalyptus ark evaluated under in vitro, revealed that Eupatorium ark was most effective with complete mycelium inhibition at 10 per cent concentration being followed by Jeevamrit with 88.78 per cent mycelial inhibition at 25 per cent concentration. Under in vivo conditions propiconazole gave maximum disease control (79.85%) however, among non-chemical components T. koningii (DMA-8), Eupatorium ark and plant extracts of Eucalyptus provided 53.99, 68.15 and 59.98 per cent disease control, respectively over check. Under field conditions (KVK Berthin & RSS Akrot), individually among chemicals three sprays of propiconazole 25EC and among non-chemical three sprays of Eupatorium ark were found most effective with 63.96 and 28.03 per cent disease control, respectively. However, integrated treatments of chemical with non-chemical, two sprays of propiconazole 25EC + one spray of Eupatorium ark resulted best with 54.10 per cent disease control whereas, single spray each of propiconazole 25EC, Eucalyptus extract and Eupatorium ark at 10 days intervals also gave good management of disease i.e., 44.37 per cent disease control over check.ThesisItem Open Access High resolution mapping of Co-Ind gene from common bean landrace KRC-5 possessing resistance to Colletotrichum lindemuthianum races(CSKHPKV. Palampur, 2020-07-07) SHABNAM; Rana, S.K.The common bean landrace KRC-5, native to Kinnaur region of Himachal Pradesh is known to possess resistance against different races of Colletotrichum lindemuthianum. In this study, an attempt has been made to fine map the resistance locus in common bean landrace KRC-5 and to convert RAPD marker linked to the resistance gene into specific SCAR marker. In addition, the effectivity of the R-gene was tested against 24 physiological races of C. lindemuthianum. The segregation studies on F2 and F2:9 mapping population obtained from Jawala x KRC-5 cross against races 3 and 211 showed a good fit of 3R:1S and 1R:1S ratio, thus confirmed the presence of a single dominant gene in landrace KRC-5. To fine map the gene, a set of 576 molecular markers comprising of SSRs and RAPDs was used for polymorphism survey. One hundred and thirty six markers found polymorphic on parents failed to distinguish the resistant and susceptible bulks in bulked segregant analysis (BSA). Therefore to accomplish the fine mapping, ddRAD sequencing based strategy using 40 RIL individuals including parents was applied to identify SNP based markers. From ddRAD 1,15,578 SNP markers were identified on 11 common bean linkage groups and finally 1,979 SNPs were retained for linkage map development using JoinMap software. In addition 28 anchored SSRs and two SCARs were also genotyped on 40 individuals (RILs & parents) used for ddRAD to validate the linkage of identified SNPs on bean chromosomes. The genotyping of 219 F2:9 RIL individuals with SCAR marker ScOPR15408 and previously developed SCAR (ScOPF6522) showed their linkage with R-gene at 2 cM distance on the opposite sides of Rlocus. Linkage map developed by using 2,009 markers including 1,979 SNPS, 28 anchored SSRs and 2 SCAR markers (linked to R-gene) showed the distribution of different markers on 11 linkage groups of common bean genome spanning 618 cM distance. The tentative position of R-gene (Co-Ind) present in landrace KRC-5 was marked on chromosome 10 along with two SCAR markers flanking the target gene and other SNP markers. However the exact position of R-gene can only be established after genotyping whole population with associated SNPs. In gene strength analysis, the landrace KRC-5 was found resistant to 17 races of C. lindemuthianum and hence could be effectively used in molecular breeding programmes.ThesisItem Open Access STUDIES ON VIRULENCE AND HOST RESISTANCE IN OAT- Blumeria graminis f. sp. avenae PATHOSYSTEM(CSKHPKV. Palampur, 2020-07-16) MALLANNAVARA, ANUDEEP B; Banyal, D.K.The investigation entitled “Studies on virulence and host resistance in Oat- Blumeria graminis f. sp. avenae pathosystem” was conducted at Department of Plant Pathology, CSKHPKV Palampur during 2016-2020. Powdery mildew of oat caused by Blumeria graminis D.C. (Speer) f. sp. avenae Em. Marchal is the most deleterious foliar disease of cultivated oat and severe losses of green fodder and grain yield has been reported in North Western Himalayas. Total 24 isolates of B. graminis f. sp. avenae causing powdery mildew were collected from 6 districts belonging to different zones of Himachal Pradesh. On the basis of symptomatology, the disease was identified as powdery mildew of oat and on the basis of morphological & molecular characterization, the pathogen causing powdery mildew of oat was identified as Blumaria graminis f. sp. avenae. To identify the resistant sources, 303 oat germplasm lines were evaluated under field conditions for 3 years and 11 lines viz., JPO-40, OS-10, OG-77, PLP-1, OL-1847, OL-1869, OL-1689, OL-1689-AVTSC, HFO-864, HFO125 and OL-6 were found resistant. Among 19 accessions belonging to 12 species of Avena also evaluated under field conditions and none of the accessions of any species was found highly resistant, however, OG-77 (A. sativa) and HFO-864-16 (A. byzantina) were found resistant against powdery mildew pathogen. The cultivars IG-03-203, JPO-20 and KRR-AK-06 were identified as slow mildewers on the basis of low values of AUDPC, high incubation and latent period, smaller size of colonies, less sporulation as represented by number of „conidiophores bearing conidia‟ per colony as compared to highly susceptible check HJ-8. Differential set of 11 lines viz., ADG-96, HFO-102, IG-03-213, JPO-40, OL-1847, OG-77, PLP-1, JO-11, OL-1867, UPO-212 and susceptible check HJ-8 was developed to study the pathogenic variability of B. graminis f. sp. avenae causing oat powdery mildew. Study of pathogenic variability on developed differential set grouped 24 isolates into 14 pathotypes (OMP-1 to OMP-14) and the isolates were grouped in different pathotypes as per their geographical locations. The OMP-2, OMP-7 and OMP-13 were highly virulent among all the pathotypes, which gave susceptible reaction on 9 out of 11 differential lines. Analysis of genetic diversity of 22 isolates of B. graminis f. sp. avenae using 4 polymorphic ISSR markers formed 12 groups at 98 per cent similarity coefficient indicating high genetic diversity among isolates. Both the pathogenic and molecular variability showed wide diversity among population of B. graminis f. sp. avenae in Himachal Pradesh. The inheritance of resistance was studied by makig crosses of 3 resistant lines i.e. OL-1847, OG-77 and OL-1689 with highly susceptible cv. HJ-8. In all three crosses, F1 population was found resistant to powdery mildew and F2 generation segregated into three resistant and one susceptible plant ratio, which confirmed that the resistance to oat powdery mildew was controlled by single dominant gene.
