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Characterization, population dynamics of Streptomyces spp. and management of potato scab
(Punjab Agricultural University, Ludhiana, 2019) Sabhikhi, Hardeep Singh; Hunjan, Mandeep
Scab of potato caused by various Streptomyces spp. has assumed serious proportions in certain potato growing regions of Punjab. A total of 2060 acres were surveyed during this study, out of which 490 acres were having scab infection. Maximum disease severity was observed in Kapurthala (7.9%), followed by Ludhiana (6.3%) and Jalandhar (6.2%) districts. During the surveys, it was observed that common scab type of symptoms were the most prevalent (89.7%), however disease induced by multiple type symptoms was viz., pitted or raised corky lesions was more severe. One hundred seven isolates of Streptomyces were collected, and characterized biochemically, pathotypically and genetically. All the Streptomyces spp. isolates could utilize hexose sugars, but not pentose sugars and most of the isolates produced melanoid pigment, an important virulence factor of this bacterium. Multiple antibiotic resistance was observed in 20 isolates. Pathotypic bioassay of Streptomyces spp. isolates revealed that Kufri Pukhraj had the highest virulence frequency (81.3%) while K. Himsona had the least (40.2%). Ninety six isolates were confirmed pathogenic. The 16srDNA amplification confirmed the identity of the genus, whereas sequence analysis of nine representative isolates revealed that Strep101 belonged to Streptomyces acidiscabies, Strep102 to reticuliscabiei, while Strep131, Strep137, Strep143, Strep145, Strep154, Strep170 and Strep173 to scabiei. All isolates were further characterized on the basis of pathogenicity genes, txtA and nec1.The txtA gene from Punjab isolates either matched with S. scabiei or S. turgidiscabies No txtA amplification was observed in S. acidiscabies, Strep101 and S. reticuliscabiei, Strep102. The nec1 gene was amplified in 84 out of 96 isolates and no nec1 was detected in S. reticuliscabiei, Strep102. Genetic fingerprinting carried out by using 17 RAPD primers clustered these into eight genetic groups. Many genetic groups had multiple virulence groups and vice-versa. To identify resistant donors, 41 potato germplasm lines were screened using representative isolates of different virulence groups. Most of the lines were susceptible to moderately susceptible. Three crop rotations viz., fallow-rice-potato, summer moong-rice-potato and sunhemp-rice-potato showed maximum per cent decrease in the population of Streptomyces spp. in soil and scab severity on potato tubers. Using disease free seed tuber or early harvesting of potato from scab sick field also observed that to reduce scab effectively. In vitro and in vivo evaluation of different chemicals against S. scabies showed that Streptocycline, streptomycin, boric acid and copper sulphate were effective. However, tuber seed treatment with chemicals viz., Kocide@0.2% and Streptocycline@100 ppm and antagonist Streptomyces spp. strain S25 and S27 had negative impact on the germination of potato seed tuber. For the management of scab, complete package involving use of tolerant cultivars, disease free seed or seed treatment, and cultural practices have to be followed.
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Over summering behaviour of Puccinia striiformis f. sp. tritici Erikss., the incitant of yellow rust of wheat and its management
(Punjab Agricultural University, Ludhiana, 2019) Tak, Parminder Singh; Pannu, P.P.S.
Yellow rust of wheat was found to survive in off season on main crop, self grown plants, ratooned plants and abandoned un-harvested plants/ fields in nine regions viz. Janjehli and Rohanda (Mandi), Bhalyani and Pulga (Kullu), Galu (Shimla), Haripurdhar (Sirmaur), Keylong in H.P. and Kargil and Leh in J&K from April to October. Aeciospores of rust from barberry caused chlorotic specks on susceptible wheat cultivar PBW 343, but no further sporulation was observed even after one month. Average minimum temperature influenced incubation period, latent period and disease severity. Incubation period increased to 19 days and 14 days with minimum average temperature above 12˚C and below 6˚C, respectively even when RH was around 90%. Minimum temperature in the range of 7-13˚C, relative humidity ≥ 90%, frequent rains during Feb-March along with winds are congenial for development and spread of yellow rust. No infection developed on barberry under in situ conditions when inoculated with teliospores from wheat. Twenty nine polymorphic SSR primers revealed polyphyletic origin of the races of Pst. Bhutti and Bhalyani (Kullu) isolates grouped together with Pst 46S119 isolate from Santokhgarh adjoining Punjab. Isolates from Sundarnagar (H.P.) grouped with Pst isolate from Fatehgarh Sahib (Punjab). Primer pair Scaffold233-153774 and Scaffold904-62635 were found to differentiate Pst 46S119 and 110S119, respectively. Barberry rust isolates were not amplified by any SSR primers specific to Pst, indicating them to be different from Pst. Early sowing (October) of wheat resulted in highest disease severity and highest area under disease progress curve as compared to November sowings. Out of four wheat varieties tested, PBW 343 was found to give highest AUDPC of upto 2041.7. Fungicides viz. Amistar top 325SC, Amistar xtra 280SC, Custodia 320SC, Opera 175SE and Tilt 25EC @ 0.1%, Nativo 75WG @ 0.6% and Taqat 75WP @ 0.3% were found to give almost complete control of yellow rust along with significant increase in thousand grain weight (26.0 to 30.3 %) and increase in yield varying from 33.3 to 40.2 per cent. Amistar top 325SC reduced the average plant height, but recorded maximum tillers in both the years without any adverse effect on yield. Maximum activity of PAL, PPO and POX was observed in resistant variety PBW 725 as compared to susceptible cultivar HD 2967. Enzyme expression under challenged conditions was more than un-challenged. Highest PAL activity was observed in Nativo, PPO in fermented butter milk and POX activity in Opera, where elevation of 10.8, 1.8 and 5.6 fold, respectively was observed as compared to unchallenged conditions. Timely sowing, varietal diversity, cultivation of resistant/ tolerant varieties, monitoring of crop during December – January, management of initial foci of infection in disease prone areas and timely advisories based on weather forecasting has been found the best strategy for management of yellow rust in Punjab.
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Characterization of Phytophthora species associated with citrus foot rot and its management in Punjab
(Punjab Agricultural University, Ludhiana, 2019) Yesmin Kaur; Thind, S.K.
Foot rot of citrus caused by Phytophthora spp. is major threat to flourishing citrus industry across the world. During surveillance, maximum disease incidence was recorded in Fazilka and minimum in Sangrur districts of Punjab. Among different agro-climatic zones of Punjab, maximum incidence was recorded in arid-irrigated zone and minimum in central zone. The disease incidence was more on Grapefruit and less on Kinnow cultivars of citrus. Oat meal agar medium was best for growth of Phytophthora. The causal agent of the disease was identified as Phytophthora nicotianae on the basis of morphological and molecular characters. Phytophthora survival was highest in irrigated and inoculated soil planted with Rough lemon seedlings and diminished in unirrigated bare soil after 15 months of inoculation. The population was maximum at 0-8 inches depth and was positively correlated with the plant root density. Maximum population was recorded in the months of July-September with temperature of 28.0-32.5 °C and relative humidity of 71.5 to 75.0 per cent. Leaf baiting was found best for detecting Phytophthora population after 24 hours with 1:1 soil and water ratio. A set of seventeen SSR primers generated 67 to 100 per cent polymorphism. Metalaxyl resistant isolate of Phytophthora showed reduced sensitivity to Ridomil Gold 68 WP at 1, 10 and 50 μg/ml with ED50 value of 30.0 μg/ml. Ridomil Gold 68 WP, Profiler 71.1 WG and Curzate M8 72 WP were found effective in vitro and in vivo against Phytophthora. Profiler 71.1 WG and Curzate M8 72 WP were also effective against metalaxyl resistant isolate.
Open Access
Epidemiology of pea rust (Uromyces viciae-fabae) and search for resistance sources
(Punjab Agricultural University, Ludhiana, 2019) Simranjit Singh; Simranjit Singh; Amarjit Singh; Amarjit Singh
Pea (Pisum sativum L.) is the second most important worldwide grown pulse crop. It suffered from many biotic and abiotic stresses but, the rust caused by Uromyces viciae–fabae (Pers.) J. Schrot is considered as one of the most important diseases of pea. The disease was prevalent in all the pea growing areas of Punjab but, sub-mountainous region was suffered more. The highest disease severity was recorded at village Burey Rajputan of Hoshiarpur district and it was identified as hot spot. A yield reduction of more than 60 per cent coupled with 11 per cent reduction in 1000 seed weight was observed under artificial epiphytotic conditions. The disease severity showed a positive correlation with maximum temperature, minimum temperature and rainfall while a negative correlation was found with morning and evening relative humidity. Early sown crop i.e. 30th September faced lower disease pressure whereas late sown crop i.e. 15th November succumbed to high disease pressure. The velocity of rust spread was observed @ 0.83 m/week during 2016-17and 0.89 m/week during 2017-18 whereas the intensity of disease spread was found more towards the south western side as it influenced by wind direction. Only three plant species (one weed i.e. Euphorbia helioscopia and two crop plants viz. Vicia faba and Lens esculentus) out of 11 were able to express the disease symptoms. A total of 128 genotypes/lines were screened for rust resistance and only a single genotype Nepal showed resistant, 34 moderately resistant, 54 moderately susceptible, 31 susceptible and 8 showed highly susceptible disease reaction against pea rust. The disease severity showed highly positive correlation with most of the plant factors under consideration but showed negative correlation with plant height. The genotypes having spreading and dwarf type growth habit were more prone to disease. All the tested chemical fungicides showed effective disease control but, 3 applications of tebuconazole were found most effective in terms of minimum disease severity and maximum green pod yield.
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DEVELOPMENT OF EFFECTIVE STRAINS OF Trichoderma SPECIES FOR THE MANAGEMENT OF FOOT ROT OF CITRUS
(Punjab Agricultural University, Ludhiana, 2019) Choudhary, Ajay Kumar; Narinder Singh
Foot rot of citrus is caused by Phytophthora spp. and is one of the most important disease of kinnow crop. Soil samples were collected from foot rot infected citrus orchards from Abohar and Fazilka areas. Three isolates of Phytophthora spp. were isolated by using leaf-disk baiting on PARPH-CMA medium and were characterized morphologically and at molecular level. For molecular analysis genus (Ph2–ITS4) and species (Pn5B–Pn6) specific primers were used and all three isolates were identified as Phytophthora parasitica var. nicotianae. Soil samples from healthy citrus orchards of Abohar and Fazilka regions were collected and total 25 isolates of Trichoderma were isolated and screened in vitro for antagonistic activity against Phytophthora parasitica var. nicotianae. Among the twenty five isolates, T20 isolate had maximum mycoparasitism, volatile and non-volatile metabolites activity i.e. 74.81 per cent, 65.61 per cent and 75.93 per cent, respectively. T20 parent isolate also had maximum cellulase and gulcanase activity i.e. 1.98 unit/ml and 1.31 unit/ml respectively. Seven parent isolates of Trichoderma (T2, T3, T4, T16, T20, T21 and T25) were selected on the basis of superiority in bioassay and biochemical studies. Molecular identification was done by using ITS1 and ITS4 universal primers and sequencing of all the seven parent isolates of Trichoderma revealed six isolates as Trichoderma asperellum and one as Trichoderma harzianum. These seven parent isolates were used to develop seven mutant strains of Trichoderma through different mutantgens like EMS (Ethyl Methane Sulfonate) and UV rays. The enzymes production was increased significantly for both glucanase and cellulase from 2.6 to 4.3 and 2.03 to 3.05 times, respectively as compared to parents strains that lead to increased antagonistic activity against Phytopathora parasitica var. nicotianae under in-vitro and vivo conditions. T20 mutant (Trichoderma asperellum) isolate had maximum mycoparasitism, volatile and non-volatile metabolites production i.e. 83.70, 79.26 and 81.11 per cent respectively. Among all the Trichoderma isolates, T20 mutant (T. asperellum) showed maximum enzyme activity of 3.62 unit/ml and 6.19 unit/ml for β-1,3-glucanase and β-1,4-glucanase enzyme, respectively. These seven parent and mutant isolates of Trichoderma were tested against foot of citrus pathogen i.e. Phytopathora parasitica var. nicotianae under green house and field conditions. All the Trichoderma mutant isolates performed better than their respective parents under green house and field conditions. T20 mutant (Trichoderma asperellum) isolate was the best and had minimum diseases incidence (13.33) per cent, maximum germination percentage (93.30), feeder root density (0.023 cm3) and recovery of lesion size (95.82 per cent). Maximum number of fruit and yield per plant was also noticed in T20 mutant (Trichoderma asperellum) i.e. 988 and 164 as compare to control which was 500 and 87kg. Cloning and Sequencing of cellulase (1, 4 β-glucanase) gene from T20 T. asperellum (parent and mutant) and T16 T. asperellum (parent and mutant)) exhibited 98 per cent homology to the endo-β-1,4-glucanase genes from T. asperellum KJ188168.1. Study of shelf life of bioformulations revealed that antagonists T. asperellum T20 (parent and mutant) and T. asperellum T16(parent and mutant) can remain potent at ambient temperature for 300 days, while at freezing temperature storage (0oC) these can remain potent up to one year.
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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.

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