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Epidemiological studies on Alternaria blight of marigold
(Punjab Agricultural University, Ludhiana, 2018) Mailem, Y.S; Amarjit Singh
Marigold (Tagetes erecta L.) is an important multipurpose flower crop grown throughout India. Its production is affected by various biotic and abiotic factors of which Alternaria blight caused by Alternaria tagetica plays an important role in economic production. The pathogen affects almost all parts of the crop under severe condition. It was found that the disease affects plant at all stages of growth with increased severity in older plants compared to younger plants. The optimum inoculum load of the fungus for disease development was 4 x 104 conidia/ml of water, although incidence and severity increased with increased inoculum load. Leaf wetness duration and temperature were also found to play a role in disease development. Temperature of 25± 1oC and wetness period of 12 h was found to be optimum creating high disease pressure. Marigold seeds were found to play role in the transmission of the disease when naturally and artificially infected by the pathogen. The pathogen also showed survivability under different conditions. Maximum survival was observed under room conditions where there were no soils during storage. Progression of disease was more towards South-eastern and North-western direction due to flowing of North-westerly and South-easterly winds during the two crop seasons. Among twenty-one marigold cultivars evaluated, four cultivars viz. Pusa Arpita, Double Dwarf Lemon, Harmony Boy and French Banita were found to be resistant under field conditions. All other remaining cultivars, except Pusa Narangi Gainda were found to be moderately susceptible. Morphological characters of marigold plant were found to show non-significant role in disease development. Biochemical analysis of defense related enzymes viz. peroxidise, polyphenol oxidase and phenylalanine ammonia lyase among eight cultivars showed that these enzyme activities were greatly reduced in susceptible marigold cultivar in comparison to resistant and moderately susceptible cultivars.
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Identification, characterization and management of major virus(es) associated with cucurbits in Punjab
(Punjab Agricultural University, Ludhiana, 2018) Dhkal, Manmohan; Sharma, . Abhishek
Viral disease was reported to be major constraint in the cucurbits cultivation under Punjab conditions. Disease survey conducted during February-August 2017 and 2018 in different cucurbits growing areas of Punjab revealed maximum viral disease incidence in district Pathankot (100%) during 2017 and Ludhiana (36.5%) during 2018. During survey blistering, yellows, leaf curl, mosaic, puckering and necrosis were observed as common viral symptoms on cucurbits. Among these, yellows and leaf curl were most prevalent symptoms on cucurbits in different districts of Punjab, whereas watermelon showed necrotic symptoms. Musk melon samples with yellows and leaf curl symptoms showed seronegative reaction against antisera of commonly occurring viruses and was found positive in PCR for whitefly transmitted begomovirus infection. Musk melon and tinda were identified as new host of begomovirus in India. Tomato leaf curl New Delhi virus (ToLNDV), Tomato leaf curl Palampur virus (ToLCPMV) and Seena leaf curl virus were characterized from musk melon showing yellows and leaf curl symptoms. ToLNDV infection was found to be associated with tinda and cucumber causing yellows and leaf curl symptoms. In watermelon, Groundnut bud necrosis virus (GBNV) was serologically detected with samples showing necrosis symptoms. Among different methods of transmission ToLCPMV associated with yellows and leaf curl symptoms was found to be transmitted only by whitefly, whereas one percent seed transmission of necrosis symptoms caused by GBNV was observed in watermelon. Out of 75 melon accessions SM/IC-267375 and WM-11 showed high resistance response against ToLCPMV in artificial screening as no symptom observed even after 45 days of sowing as compared to susceptible checks. Three watermelon accessions viz., WM-6-2-2-1-1-2, WM-36 and WM-829833 produced mild necrotic symptom during natural screening. Among different natural products used for management of viral disease in musk melon, buttermilk was found to be highly effective at 20% concentration with least per cent disease incidence (50.8%) as compared to 10 % (64.2 %) and 5 % (71.1%) concentrations. Besides buttermilk, kaolin was also found to be effective in the management of viral diseases of musk melon. Among different dates of sowing least incidence of viral disease was observed on late sown or transplanted musk melon crop 15 March (38.96%) followed by the 5 March (46.7%) sown crop. Among different methods of planting, tray transplanted musk melon crop showed less incidence (48.3%) compared to directly sown crop (50.4%) whereas, normally transplanted crop showed maximum disease incidence of 55.7 per cent.
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Molecular and biochemical bases for resistance to sclerotinia stem rot in introgressed Brassica juncea lines
(Punjab Agricultural University, Ludhiana, 2018) Gill, Rupeet; Sandhu, Prabhjodh Singh
Sclerotinia rot caused by the fungus Sclerotinia sclerotiorum (Lib.) de Bary is one of the most serious and damaging diseases of oilseed Brassicas and there is keen interest worldwide to identify Brassica genotypes with resistance to this pathogen. In this study, introgression lines derived from hybridization of wild crucifers with Brassica juncea were evaluated for response to stem rot. These were challenged against local Sclerotinia sclerotiorum isolate and 24 lines giving variable reaction were selected. Apart from this a total of 58 isolates of S. sclerotiorum were collected from different Brassica growing states (Punjab and adjoining states). These were evaluated for their pathogenicity on susceptible B. juncea cultivar RL1359 and based on disease progression; they were divided into five groups. Further 24 isolates were chosen from these groups and analysed for biochemical parameters. It was found that oxalic acid production significantly correlated with the pathogenicity of the isolates and lesser/no correlation was observed for pectinase enzymes. These 24 isolates were then used to further evaluate the selected 24 introgression lines. The observation on progression of lesion length revealed a variable response of lines to different isolates. Disease scoring was done and thus lines were categorised as resistant, moderately resistant and susceptible. Qualitative analysis was carried out on lines from different categories for genes NPR1, COI1 and EIN2. Upregulation in COI1 gene after fungal infection in resistant lines indicates the possible induction of jasmonic acid mediated defence response. Four lines from each category were selected for biochemical analysis involving SOD, POX, PAL, total phenols and lignin. All of these had a negative correlation with disease and the genotype with a greater level of resistance was superior to the susceptible one in regard to phenol and lignin production and the activities of defence enzymes. These results suggest that a combination of these defence responses in Brassica may contribute to greater plant resistance to S. sclerotiorum and that these enzymes have potential use in selection of resistant genotypes.
Open Access
Evaluation of induced systemic acquired resistance against oomycete pathogens in potato and muskmelon
(Punjab Agricultural University, Ludhiana, 2017) Astha; Sekhon, P.S.
Oomycetes pathogens; Phytophthora infestans causing late blight of potato and Pseudoperonospora cubensis causing downy mildew in muskmelon are most important foliar diseases, causing significant yield losses. The present study was conducted to reduce fungicide load and work out alternate method for control of these two diseases. Different SAR compounds were tested and exogenous foliar sprays of different conc. of Salicylic acid, Jasmonic acid and Bion (Benzothiadiazole-BTH) @ 50µM, 250µM,500µM, 1000µM and of Beta amino butyric acid of 20 mM, 30mM, 50 mM, 100mM were given for inducing resistance in potato against late blight and in muskmelon against downy mildew. Twenty germplasm lines/varieties of potato and thirtythree germplasm lines/varieties of muskmelon were tested with these four compounds in year 2014, 2015 and 2016. Five lines of potato and eleven lines of muskmelon showed moderate resistance along with better response to SAR elicitors against oomycete pathogens. Concentration of Salicylic acid, Jasmonic acid and Bion @ 500 µM, and Beta amino butyric acid @ 50 mM gave good control of disease. Salicylic acid gave best results with 81.28 per cent disease control followed by Jasmonic acid with 76.23%; whereas Bion and Beta amino butyric acid were almost at par with each other and gave 74 % disease control as compared to control plants. Recommended spray schedule of fungicides gave 93 % disease control, which was at par with disease control given by Salicylic acid and BABA spray schedules in combination with contact fungicides. Protein content of treated muskmelon plant varied from 10.5 to 13.1 mg/g fresh weight compared to 5.4 mg/g fresh weight in control. Similarly, treated potato plants showed 6.4 to 7.7 mg/g fresh weight total protein as compared to 4.0 mg/g fresh weight in control. Induction of proteins and defense enzymes was systemic in nature in response to all the four elicitors. Electrophoretic protein profiling of treated potato and muskmelon plants also confirmed the induction of pathogenesis-related proteins ranging from 15- 75 kDa along with some other proteins. The inducers also stimulated enzymatic activities i.e. β-1,3 glucanase, Peroxidase (POD), Polyphenol oxidase (PPO), Phenylalanine ammonia lyase (PAL) from 26 to 99 % indicating induced resistance in treated potato and muskmelon plants as compared to control. Total chlorophyll and carotenoids also showed spike of 2% to 91 % in response to elicitors. Thus integration of disease tolerance and SA spray schedule resulted in effective and economical control in potato against late blight and in muskmelon against downy mildew.
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.
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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, Narinder
Sheath 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.
Open Access
Variability in Ustilaginoidea virens (Cke.) Tak. causing false smut of rice and identification of resistance sources
(PAU, 2014) Rani, Ritu; Sharma, Vineet K
False 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.
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 Mohan
Disease 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/hill
Open Access
Investigations on foot rot (Fusarium moniliforme Sheld.) of basmati rice with reference to survival and variability of pathogen and host-resistance
(Punjab Agricultural University, Ludhiana, 2017) Puyam, Anita; Pannu, P.P.S.
The foot rot (bakanae) disease of rice caused by Fusarium moniliforme (syn. F. verticilloides) has become a serious bottleneck in the successful cultivation of basmati rice in Punjab and its adjoining areas. Studies of cultural characteristics of F. moniliforme on Potato dextrose agar (PDA), Soil Extract Agar (SEA) and Spezieller Nahrstoffarmer Agar (SNA) showed that all the 38 isolates produced whitish to pinkish color on the surface with dense and fluffy growth on both PDA and SEA, and moderate but not fluffy on SNA. The best growth was observed on PDA, followed by SEA and SNA. The growth rate of different isolates varied between 12.28-25.86-mm/ 48 hrs on PDA, between 9.09-19.59 mm/ 48 hrs on SEA and between 5.97- 16.44 mm/ 48hrs on SNA media. The size of the spores varied in the range of (6.8-9.8) X (2.9-6.0) µm for microconidia with no septa and (24.23-62.97) X (2.90-4.27) µm for macro-conidia having 3-5 septa. Micro-conidia were formed in chains on water agar with KCl and none of the isolates produced chlamydospores. DNA of 35 out of the 38 isolates was amplified with the set of primers VERT1 and VERT2 specific for F. verticilloides and gave a single amplicon of 800bp. All the isolates showed variable production of gibberellic and fusaric acid. All 38 isolates produced gibberellic acid whereas fusaric acid was produced by 30 isolates. Statistical analysis showed that the production of fusaric acid significantly correlated positively with the number of stunted plants and gibberellic acid production positively correlated with the number of elongated plants. Molecular studies using Simple Sequence Repeats (SSR) primers revealed 102 alleles using 27 primers. Out of 27 markers, all of the markers showed polymorphism with least polymorphism shown by primer (8H01) and no polymorphism by primer 2H06. Maximum number of alleles was produced by the primer 5H09. Polymorphic Information Content (PIC) values varied from 0.10-0.89 with an average of 0.46. Eleven SSR primers revealed PIC values higher than the average. The primers 7H05, 11H01, 5H09, 3H19, 10H09, 10H01, 9H05, 1H02, 2H17, 2H15, 3H02, 11H03 and 10H07 showed PIC values equal to or higher than 0.50. The genetic dissimilarity index ranged from 25 to 75%. The variability in pathogen, its survival and identification of resistant sources will help to formulate effective strategies for management of foot rot. Investigations on survival of Fusarium moniliforme in soil revealed that the pathogen doesn’t survive in soil up to next season. Out of nine weeds evaluated against foot rot under artificial inoculation conditions in field, none of the weeds was infested. Out of 134 basmati germplasm lines screened under artificial inoculation field conditions, 24 lines were moderately susceptible, 4 lines were susceptible, 4 were highly susceptible, 27 were moderately resistant, 7 lines were resistant and 5 lines were highly resistant.

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