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Biochemical defense response and genetic basis of resistance against Ascochyta blight (Ascochyta rabiei (Pass.) Labr.) in chickpea (Cicer arietinum L.)
(Punjab Agricultural University, 2024) Abassy, Omer; Dr Upasana Rani
Chickpea (Cicer arietinum L.), the second-largest global pulse crop, is under threat from highly variable nature of Ascochyta rabiei, causing widespread disease damage under favourable environmental conditions. This disease, Ascochyta blight is a major concern worldwide, with insufficient resistant sources and frequent breakdowns due to rapid pathogen evolution. This study evaluated 238 kabuli chickpea genotypes for resistance to Ascochyta blight (AB) over the growing seasons from 2019-2022 at Punjab Agricultural University under artificial epiphytotic conditions, identifying 18 resistant lines (GLK 10-40, GLK 20054, GLK 20055, FLIP 10-298C-IFC-S2, FLIP 10298C-IFC-S1, CS-3-E-24, FLIP 09 256C-55, FLIP 07-314C-57, FLIP 10-243C, FLIP 09-194C, FLIP 08-104C, FLIP 04-219C, ICCV 55233, ICCV 55215, ICCV 55135, ICCV 55108 and ICCV 155141) belonging to different genetic backgrounds with disease scores of 1.0 to 3.0, whereas seventy-three (73) lines demonstrated a moderate level of disease resistance with a disease rating of 3.1-5.0. The activities of defense-related enzymes (PAL, TAL, PPO, POD) associated with phenol metabolism along with lignin and total phenol content were compared among six kabuli chickpea lines, viz., five resistant (GLK 10-40, GLK 20055, FLIP-09-194C, FLIP-04-219C, ICCV 55215) and one susceptible (GLK 17301) treated lines that exhibited differential responses to Ascochyta blight at tested time intervals of 48, 96, 144 and 240 hr post-inoculation respectively. The perusal of data showed increased activity of all enzymes (PAL, TAL, PPO, POD) till 96 hr after inoculation, whereas the non-enzymatic contents such as lignin and total phenols showed the maximum enzyme activity till 144 hr after inoculation. However, enzyme and non-enzymatic activity remained almost constant with no significant difference in all control/untreated resistant and susceptible line(s) at different time intervals. For understanding the genetic basis of resistance, the F2 population comprising of 162 plants derived from the cross, GLK 24096 (resistant) and L 556 (susceptible) was assessed under artificially epiphytotic conditions against local isolate of Ascochyta rabiei. Of 162 plants evaluated, 39 plants were grouped in resistant (R ) category (disease score ≤ 5.0 ) and 123 were classified as susceptible (S) with score of five and above. Consequently, the F2 plants exhibited a segregation ratio of 1R:3S, suggesting that a monogenic recessive gene governed resistance to Ascochyta blight. Of thirty four SSR markers associated with Quantitative Trait Loci (QTLs)/genes were used for confirmation of resistance in identified eighteen resistant and six susceptible lines, only 19 SSR markers depicted polymorphism. Notably, SSR markers, STMS11, TA194, TS12, TA146, and TA2 exhibiting significant polymorphism, indicated tight linkage to Ascochyta blight resistance. Phylogenetic analysis showcased distinct clustering based on resistance, offering insights into the genetic basis of resistance. These findings informed about targeted resistance breeding and sustainable disease management with defence-related enzymes/identified markers holding promise for developing superior, resistant and resilient chickpea varieties contributing to crop sustainability and productivity.
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Understanding sunflower - Macrophomina phaseolina interaction under water stress conditions
(Punjab Agricultural University, 2023) Navkiran Kaur; Sharma,Pankaj
A total of 35 isolates of Macrophomina phaseolina causing charcoal rot were collected from eleven kharif and rabi crops from different districts of Punjab and adjoining states. Macrophomina phaseolina isolated from sunflower field, Ludhiana was characterized on cultural and morphological and molecular basis. The isolates infecting sunflower showed highest sequence homology of ITS genomic region with M. phaseolina isolate from Indore, Madhya Pradesh NCBI accession no. MT127404. This isolate was further used to standardize the inoculation techniques under field and polyhouse conditions. Tooth pick inoculation technique was found most effective for large scale screening while sorghum grain inoculation method was proficient under polyhouse conditions. A set of 71 sunflower genotypes were evaluated using tooth pick inoculation technique against charcoal rot under irrigated and restricted irrigation conditions. Four genotypes viz; 75B, EC6078261, OPH137, and OPH 172 were found moderately resistant under irrigated conditions whereas only two genotypes 75B and EC6078261 showed moderately resistant reaction in restricted irrigated conditions. The present study was first attempt to investigate the water stress-Macrophomina phaseolina interaction on different components of physiological, plant growth and yield parameters along with disease incidence. A significant reduction in total chlorophyll content (54.78 %), relative water content (47.06 %), membrane stability (51.75 %), sugar (69.00 %), protein (13.90 %) and proline (56.66 %) content was observed with inoculum density of 4 g/kg soil at soil moisture levels of 40 % and 60 %. The inoculum density of 4 g/kg of dry soil resulted in maximum mortality during seedling stage and growth period. Thus, varied inoculum density and water stress conditions made the sunflower plants more prone to dismissive physiological alterations. It led to biomass reduction viz; root length (41.27 %), root width (76.74 %), root weight (85.37 %), shoot length (56.71 %) and shoot weight (79.06 %). Thus it resulted in per plant seed yield reduction (95.45 %) at 52.62 to 100 % disease severity at higher inoculum density (4g, 6g, and 8g/kg dry soil). The expression levels of the defense related genes LOX, ACCO1, PAL, SOD, and APX were also modified under varying degree of water stress and inoculum density. The strong correlation between low soil moisture and disease severity implies that the proper irrigation of the field can be suggested as cultural management practices for the farmers.
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Management Of Sclerotinia Stem Rot Of Rapeseed-Mustard Incited By Sclerotinia Sclerotiorum (Lib.) De Bary
(Punjab Agricultural University, 2023) Yadav, Monika; Sandhu, P.S.
Sclerotinia stem rot caused by Sclerotinia sclerotiorum, is an important fungal disease of rapeseed and mustard crop because of its destructive nature. In the present study, 112 genotypes of rapeseed-mustard screened against stem rot disease. Ten genotypes (EC223400, EC765482, IC268306, IC296703, IC491169, IC491178, IC491197, IC491290, IC531377 and IC538699) were found moderately resistant to the pathogen. Early sowing of brassica during first week of October results in lower incidence of the disease and higher yield compare to the late sowings. Poison food technique was used to evaluate botanicals and fungicides under in vitro condition. Among the botanicals maximum per cent inhibition in mycelial growth was observed in case of Chenopodium album at 20 per cent concentration. Whereas, in case of fungicides complete inhibition (100%) was observed in combination fungicides picoxystrobin 7.05%+ propiconazole 11.7% w/w SC, azoxystrobin18.2% w/w + difenoconazole 11.4% w/w SC and fluopyrum 17.7% w/w + tebuconazole 17.7% w/w SC at a concentration of 5, 10 and 25 ppm, respectively. Under in vitro conditions, combination fungicides were more effective against S. sclerotiorum compared to contact fungicide mancozeb 75 % WP which resulted in lower inhibition of pathogen growth even at higher concentrations. The same set of botanicals and fungicides were used under in vivo conditions as a foliar spray application. Chenopodium album at 40 % concentration was observed most effective in reducing the per cent lesion length as compared to the control. Combination fungicide picoxystrobin7.05%+ propiconazole 11.7% w/w SC resulted in lower lesion length development and highest yield as compare to the control in Brassica juncea var. RLC 3.
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Biodiversity of seed-borne pathogenic mycoflora of wheat (Triticum aestivum L.) in Punjab
(Punjab Agricultural University, 2023) Rubleen Kaur; Tak, Parminder Singh
A total 105 grain markets of 23 districts of Punjab were surveyed for study of seed-borne mycoflora during 2021-22 and 654 grain samples were collected. Grains were visually inspected for incidence of Karnal bunt (KB), Black point (BP) and shrivelling. Prevalence of Karnal bunt, Black point and shrivelled grains was 10.24%, 55.20% and 79.51 per cent, respectively. Grain samples from Patiala and Mansa were found to have statistically lowest KB, black point and shrivelled seeds while highest incidence was observed in Pathankot followed by Hoshiarpur and Mohali. Three pathogenic and nine opportunistic fungi were isolated from these samples. Fungal diversity in Sarhali Kalan samples (66.67%) was highest, followed by Sirhind-1, Jalandhar, Rajpura (58.3% each). Bikhi, Nabha, Dadehra, Bucho mandi, Panjgrain (16.67%) carried low mycoflora load. All the samples collected met the minimum seed certification standards for germination. Maximum vigour index I and II of 3327.6 and 26.46 was observed in grain samples collected from Doraha-1 and Shahkot, respectively. Prevalence of Alternaria spp. (94%) was highest followed by Fusarium spp. (64%), Aspergillus flavus (48%) whereas, low prevalence was observed for Exserohilum sp. and Leohumicola sp. (2% each). The fungal isolates were characterized on the basis of morphology and cultural characteristics into 11 fungal genera belonging to 7 families which included Alternaria alternata, Bipolaris sorokiniana, Curvularia verrucosa, Curvularia spicifera and Exserohilum rostratum from Pleosporaceae family (34%); Aspergillus flavus, A. japonicus and Penicillium digitatum, from Aspergillaceae (17%); Fusarium equiseti from Nectriaceae (8%); Epicoccum purpurascens from Didymellaceae (8%); Leohumicola sp. from Incertae sedis (8%), Chaetomium globosum and Pseudothielavia terricola from Chaetomiaceae (17%) and Rhizopus stolonifer from Rhizopodiaceae (8%). Sequencing of Curvularia verrucosa, Bipolaris sorokiniana, Curvularia spicifera, Aspergillus japonicus, Pseudothielavia terricola, Alternaria alternata and Fusarium equiseti was done and cultures have been deposited in MTCC repository, Chandigarh. Thirty nine fungal isolates were inoculated on three wheat varieties and determined for seed health, foliar blights and carryover studies. Opportunistic fungi such as Curvularia sp. (isolate CUS-1), Aspergillus sp. (isolates AF-1, AF-7), Rhizopus sp. (RIZO-1), Penicillium sp. were found to reduce germination under field conditions. Highest average foliar blight severity was observed in Gurdaspur (26.98%) as compared to 20.60 per cent in Ludhiana. Bipolaris isolate BIS-1, Aspergillus flavus AF-1, Curvularia CUT-1, Alternaria ALT-6 and ALT-10 resulted high severity in Ludhiana while Fusarium FUS-1, Rhizopus RIZO-1, Drechslera DRH-3, Aspergillus flavus AF-7 and Alternaria ALT-8 were highly pathogenic in Gurdaspur. The seed inoculated with these fungi induced foliar blight, affected the germination as well as severity of foliar blight however, they could not be re-isolated in all the cases indicating their primary role only up to seedling stage. Overall, Alternaria spp. had the highest frequency in grain market samples as well as carryover studies.
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Detection, variability and translocation of Potato virus Y (PVY) infecting potato in Punjab and identification of host plant resistance
(Punjab Agricultural University, 2023) Belludi, Rakesh; Sharma, Abhishek
This study aimed to investigate the variability of Potato virus Y (PVY) infecting potato crop of Rabi season in Punjab, encompassing symptomatology, serology and molecular aspects. The study revealed prevalent symptoms, such as mild mosaic, chlorotic patches, interveinal chlorosis, puckering, veinal necrosis and stem necrosis, on potato plants caused by various potato viruses including PVY. ELISA tests confirmed the presence of six out of seven viruses in potato samples. Among them, PVX exhibited the highest incidence (78.08%), followed by PVYN (51.37%), PVM (20.55%), PVYO/C and PVS (13.01%). PLRV exhibited a lower incidence (2.74%) and no samples tested positive for PVA. Notable regional variations noticed, with Ludhiana showing high incidences of PVYN (78.43%), PVYO/C (31.37%), PVM (33.3%) and PVS (35.29%). Conversely, Kapurthala displayed a 100% PVX incidence, while Jalandhar had the highest PLRV (19.05%) incidence. In nucleic acid-based detection through RT-PCR, the primer NIb2F/3R effectively amplified the desired ~350bp amplicon in all PVYN and PVYO/C seropositive samples. Whereas, the qPVY NIb F2/CP R2 primer generated the ~1.5kb fragment in only 24 out of 67 samples, further confirmed via BLASTn sequence analysis as PVYO, PVYNTN and PVYN-Wi strains. Further virus translocation studies revealed a wide range of carryover rates, spanning from 40% to 92.46%, from infected mother plants to daughter tubers, exhibiting significant variation in viral titre across genotypes and among tubers. Spatial translocation within the tuber's root zone revealed that the bottommost tuber displayed a relatively lower PVY titre. Interestingly, freshly harvested tubers exhibited a higher viral titre at the heel end (stem end) compared to the rose end eyes in all examined tubers. After storage, a contrasting trend emerged in sprouted tubers, where the highest PVY titre was found in rose end eyes and the lowest in heel end eyes. In germplasm screening against resistance to PVY, one germplasm (KP -16-19-14) displayed high resistance (HR), while 16 exhibited resistant (R), 20 moderately resistant (MR), 22 moderately susceptible (MS), 10 susceptible (S) and two were highly susceptible (HS) to PVY. Following PVY inoculation, significant reduction was observed in chlorophyll fluorescence parameters, viz., Fv/Fm, Y(II), qP, and qL values in susceptible cultivars, accompanied by notable increases in NPQ, Y(NO) and qN values. Susceptible genotypes displayed yield losses ranging from 13.64 to 58.84% with viral titre ranging from 0.014 mc to 419.673 mc. Finally, a farmer-friendly and cost-effective vertical flow assay (VFA) was developed for rapid PVY detection in potato leaf samples. The sensitivity of the developed assay was equivalent to commercially utilized ELISA. Specificity test confirmed assays precision for prevalent PVY strains and its non-specificity to other potato viruses. Developed assay exhibited 95% accuracy compared to RT-PCR.
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Characterization of Fusarium species associated with pokkah boeng disease of sugarcane
(Punjab Agricultural University, 2023) Meena, Manisha; Anuradha
Sugarcane (Saccharum spp. hybrid complex) is an important cash crop of India. Among the various fungal diseases, pokkah boeng caused by Fusarium spp. has emerged as an important disease in this crop. As the occurrence of this disease has been reported from Punjab till date the cultural, morphological and molecular aspects of this disease are not studied. Keeping in view, the present research was carried to characterize the Fusarium spp. associated with pokkah boeng disease of sugarcane. Survey was conducted in the sugarcane growing command area of sugar mills (nine co-operative and seven private) of Punjab during the year 2021-22. The maximum average incidence of pokkah boeng was reported on commercially grown variety Co 0238 (17.70 %) which depicted that the said variety is highly susceptible for pokkah boeng disease. Thirty isolates of Fusarium were obtained after isolation and purification from diseased samples collected during the survey. On the basis of pathogenicity tests, all the test isolates of Fusarium found to be virulent by producing necrotic lesion in detached leaf assay and typical symptoms of pokkah boeng in syringe method. Among all the isolates, Fs23 and Fs25 were highly pathogenic. The isolates showed significant variations on basis of their cultural and morphological characteristics and were categorized into various groups. Molecular study of isolates revealed that isolates Fs7, Fs20, Fs27, Fs29 showed 100 per cent similarity with the ITS, RPB2 and TEF-1α gene sequences of F. sacchari available in GenBank whereas isolates Fs13 and Fs17 showed 99 per cent similarity with F. proliferatum. Hence, the two species of Fusarium namely F. sacchari and F. proliferatum were identified as causal pathogens associated with pokkah boeng disease of sugarcane in Punjab. Genetic diversity estimate by seven ISSR primers showed high degree of polymorphism. Two isolates Fs16 (Jalandhar) and Fs18 (SBS Nagar) were found to be highly diverse among all test isolates. Sugarcane clones along with commercially released varieties were screened against pokkah boeng to identify the resistance sources in which 23, 18 and four clones/varieties showed resistant, moderately resistant and moderately susceptible disease reaction, respectively.
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Characterization of citrus greening pathogen(s) and its integrated management
(Punjab Agricultural University, 2023) Katoch, Sonali; Arora, Anita
Surveys conducted during 2019-21 to assess the distribution and intensity of greening disease in citrus orchards of Punjab revealed that its prevalence was high in the district Ludhiana (33.33%) followed by Hoshiarpur (32.60%), Fazilka (27.59%) and Faridkot (19.31%). The maximum mean disease incidence (16.60%) was observed in Hoshiarpur and the maximum mean disease index (11.98%) was observed in Ludhiana. Leaf mottling, yellowing, green islands and bi-coloured fruit symptoms of disease were observed on different cultivars of citrus. Multilocus sequence typing using 16S rDNA, CLIBASIA gene locus and tufB-secE-nusG-rplKAJL-rpoBC gene cluster was followed to identify and characterize the pathogen. Three primers (OA1/OI2c, CGB F/R and Las606/LSS) corresponding to bacterial 16S rDNA sequence were used on a total of 153 samples, and positive reactions were observed with all the three primer pairs. However, CGB F/R and Las606/LSS had higher detection rates (69.9%) than the OA1/OI2c primer (37.8%), indicating more robustness of the former. Similar results were observed with nursery samples of Punjab. The root samples from greening positive plants collected in the month of February were also found positive with five primer pairs (OA1/OI2c, CGB F/R, rplJ/K F/R, Las606/LSS and LapGP F/R) whereas, the root samples collected in May and September months showed no amplification. In addition, the CLIBASIA gene locus based primer pairs corresponding to sequences coding for hypothetical, heamolysin and type-I secretary system protein and one primer based on tandem repeats were used on 15 isolates of greening bacterium. All the samples showed positive reaction with five primer pairs. The disease was characterized by sequencing of 16S rDNA and CLIBASIA gene locus from five isolates with CGB F/R, three with Las606/LSS, two clones with OA1/OI2c and four with CLIBASIA gene locus which showed 99-100% similarity with reported isolates of Candidatus Liberibacter asiaticus (Ca. Las) from Asia. The phylogenetic analysis of these isolates showed 99-100% similarity with Ca. Las. The isolation of Ca. Las from leaves of PCR positive Kinnow plants was attempted on Liber A media, but it could not be cultured. The cultivars viz., Kinnow, Daisy, Musambi and Kagzi Lime were observed to be the best indicator plants for indexing. For integrated management of citrus greening, the treatment consisting of zinc sulphate + manganese sulphate + boric acid, tetracycline hydrochloride and 2,4-dichlorophenoxyacetic acid (2,4-D sodium salt) was proved significantly superior to other treatments in reducing the percent disease index (13.99) and providing 59.29 percent disease control with significant increase in yield (54.87 kg/tree) as compared to control (38.32 kg/tree).
Open Access
Mapping quantitative trait loci and biochemical analysis of sheath blight resistance in rice (Oryza sativa L.)
(Punjab Agricultural University, 2023) Thesiya Mayur Rajeshbhai; Lore, Jagjeet Singh
Sheath blight caused by Rhizoctonia solani Kühn, is one of the most prevalent diseases of rice in South East Asia. Use of resistant sources is an eco- and farmer friendly and more economically approach to mitigate yield losses due to this disease. The aim of the present study was to identify potential quantitative trait loci (QTLs) linked with resistance to sheath blight and associated with temporal spike in defense related enzymes as well as histopathological patterns during infection process of R. solani. Next generation sequencing (NGS) assisted bulk segregant analysis (BSA) was integrated with R package i.e. QTLseqr to map QTLs associated with sheath blight resistance in F3 mapping population derived from the cross between susceptible rice cultivar PR121 and resistant parent ShB1. The five QTLs namely qShB1, qShB3, qShB5.1, qShB5.2 and qShB6 were identified on chromosome 1, 3, 5 and 6, respectively. Activity of defense related enzymes such as phenylalanine ammonia lyase, polyphenol oxidase and peroxidase were estimated in parental lines along with three sets of progeny lines which showed significantly high activity in resistant parent as compared to the susceptible parent after R. solani inoculation. The moderately resistant (MR) lines expressed maximum enzyme activity than the highly susceptible (HS) lines. The pathogen R. solani exhibited more profuse and dense hyphal growth with close contact to surface grooves, lobate appressoria-like structures, higher number of microsclerotial development and longer lesion length on HS line as compared to the MR line. The present results provided better understanding of host-pathogen interaction and novel QTLs identified may be used in rice breeding program for the development of durable resistance to sheath blight in rice cultivars.
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Efficacy of Ty genes against begomovirus species causing tomato leaf curl disease in Punjab
(Punjab Agricultural University, 2023) Nayak, Lopamudra; Sharma, Abhishek
Tomato leaf curl disease (ToLCD) transmitted by vector whitefly (Bemisia tabaci) is major constraint in tomato production in the Indian subcontinent. During 2021-22 symptoms noticed on leaf curl disease infected tomato were yellowing, mild to severe curling of leaves, mosaic, puckering, stunting, excessive flowering and rudimentary leaves. Symptom variability was more in rainy season compared to winter season. Infected plants showed the association of tomato leaf curl NewDelhi virus (ToLCNDV), tomato leaf curl Palampur virus (ToLCPalV) and tomato leaf curl Karnatka virus (ToLCKV) along with associated satellites by PCR assay. Partial genome characterization of begomovirus from infected samples confirmed the association of ToLCNDV, ToLPalV, ToLKV from these samples. Apart from these chilli leaf curl India virus (ChiLCV), tomato leaf curl Joydebpur virus (ToLCJoV), croton yellow mosaic virus (CYVMV) along with a new species of papaya leaf crumple virus were (PLCrV) were recorded for the first time from tomato in Punjab. Plant age 20 to 40 days after sowing was found to be most susceptible stage to begomovirus infection as considerable yield loss (up to 90.4 %) was observed during inoculation of predominant begomovirus. ToLCNDV along with beta satellites was found to cause maximum yield loss compared to helper virus alone. The kinetics of virus multiplication in tomato genotype with different Ty gene(s) combinations showed accumulation of viral genomic unit and symptom severity were lower in tomato genotypes (PVB-4-1, NLLR-1) carrying Ty-1+Ty-2+Ty-3 and genotype CA-4 carrying ty-5+Ty-6 gene combination as compared to susceptible genotype Punjab Chuhhara (1179.18 x106) when inoculated with ToLCNDV+beta satellite.

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