1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Molecular diagnosis and management of Papaya ringspot virus causing papaya ringspot diseases(Department of Plant Pathology, College of Agriculture,Vellayani, 2024-04-17) Josiya Joy; KAU; Radhika, N SThe research work entitled “Molecular diagnosis and management of Papaya ringspot virus causing papaya ringspot disease” was undertaken in the Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram, during 2019-24, with the objectives; molecular diagnosis and recombinant coat protein production of Papaya ringspot virus (PRSV), and evaluation of the efficacy of beneficial microorganisms and botanical inthe management of papaya ringspot disease (PRSD). Roving survey was carried out across five Agro-ecological units (AEUs) of Kerala. The disease incidence (DI) ranged from 50.25 per cent (Kayyur-Cheemeni) to 100 per cent (Kalliyoor, Venganoor, Balaramapuram, Pallichal, Kayamkulam, Mavelikkara, Velukkara, Irinjalakuda and Shoranur). Vulnerability index (VI) of the plants to PRSV in the surveyed locations ranged from 33.54 (Badiyadkka) to 98.22 (Kalliyoor). Serological and molecular detection confirmed the presence of PRSV in all the 20 symptomatic samples collected during survey. Phylogenetic tree constructed with the deduced amino acid sequences of CP gene of 11 Kerala PRSV isolates, revealed that Thiruvananthapuram isolates clustered together, indicating their relatedness during evolution. Mechanical inoculation on two months old Red Lady papaya seedlings under insect proof conditions was carried out to identify the most virulent PRSV isolate collected from different AEUs. At three months after inoculation (MAI), Kalliyoor isolate exhibited highest VI (96.63) followed by Alur (95.48) and Venganoor (95.22) isolates. The lowest VI was observed in Kayyur-Cheemeni isolate with 57.95 VI, followed by Cherpulassery (60.58). The recombinant coat protein of PRSV was induced in pLATE 31 expression vector with C terminal histidine tag, within BL21(DE3)pLysS expression host. PRSV recombinant coat protein was purified using Ni-NTA column chromatography. A single band was observed at 35 kDa in SDS-PAGE analysis and western blotting with PRSV antiserum, confirmed the presence of purified recombinant coat protein in the soluble fraction. Pot culture experiment was conducted to evaluate the management of PRSD using Piriformospora indica. The initial colonization of P. indica inside the papaya roots was observed five days after germination of the seeds grown in P. indica massmultiplied medium. Prophylactic colonization of P. indica exhibited lowest VI (23.10) and 68.27 per cent reduction in VI over control diseased plants at five months after PRSV inoculation (MAI). Double antibody sandwich - Enzyme linked immunosorbent assay (DAS-ELISA) at 5 MAI revealed lowest absorbance (0.23) indicating lowest virus titre in P. indica pre-colonized plants upon PRSV inoculation, compared to control diseased plants (1.23). The accumulation of reactive oxygen species (ROS) i.e., H2O2 and superoxides in the leaves were analyzed using DAB (diaminobenzidine) and NBT (nitro blue tetrazolium chloride) staining respectively. P. indica-colonized plants upon PRSV inoculation indicated a higher initial accumulation of ROS at three weeks after inoculation (3 WAI) and further reduction at 6, 9 and 12 WAI of PRSV, compared to control diseased plants. P. indica-colonized plants also exhibited enhanced antioxidant defence enzyme activity viz., catalase, peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, glutamate synthase and superoxide dismutase compared to control diseased plants. Amongst PRSV inoculated treatments, P. indica pre-colonized plants exhibited highest number of leaves (20.86), leaf area (365.14 cm2), plant height (108.29 cm), stem girth (7.26 cm), shoot biomass (423.43 g), root biomass (133.96 g) and chlorophyll content (2.84 mg g-1 of fw) at five months after transplanting (5 MAT). Effect of P. indica, Bougainvillea spectabilis leaf extract (10 %) and Pseudomonas fluorescens (2 %) were evaluated against natural incidence of PRSD under field conditions at Instructional farm, College of Agriculture, Vellayani and Coconut Research Station, Balaramapuram, Thiruvananthapuram. P. indica-colonized plants exhibited lowest VI (37.63), with highest reduction in VI over control (53.85 %) followed by B. spectabilis treated plants (37.49 %) and P. fluorescens treated plants (33.31 %) at 12 months after planting (12 MAP). In DAS-ELISA, lowest virus titre with absorbance of 0.438 was observed in P. indica-colonized plants at 12 MAP, compared to the highest virus titre in control plants (1.267). B. spectabilis treated plants (0.596) also exhibited reduction in virus titre followed by P. fluorescens treated plants (0.625) at 12 MAP. P. indica-colonized plants exhibited enhancement in growth parameters viz., number of leaves (24.00), leaf area (1127 cm2), stem girth (42.74 cm) and plant height (207.39 cm) at 12 MAP. P. indica-colonized plants also enhanced the yield by 44.68 per cent followed by B. spectabilis treated plants (24.13 %) and P. fluorescens treated plants (17.99 %). Moreover, fruits from P. indica-colonized plants expressed significantly superior quality parameters. Thus, findings from the present study could aid in the preliminary detection and management of the virus, thus mitigating the widespread infection caused by PRSV. The recombinant PRSV coat protein produced in this study could be used for the development of PRSV antiserum. Additionally, our research highlights the efficacy of eco-friendly management strategies for papaya ringspot disease, with P. indica-colonization @ 106 cfu g-1 and also with four foliar sprayings as well as soil drenching of B. spectabilis leaf extract (10 %) applied at fortnightly intervals, starting from one month after planting. More field trials are to be conducted to integrate this strategy in integrated disease management (IDM) package for the effective and sustainable management of PRSD in papaya.Item Piriformospora indica and its water diffusible exudates for the management of chilli anthracnose incited by colletotrichum capsici (Syd.) butler and bisby(Department of Plant Pathology, College of Agriculture, Vellayani, 2024-07-05) Elizabeth, T Jojy; KAUThe research work entitled “Piriformospora indica and its water diffusible exudates for the management of chilli anthracnose incited by Colletotrichum capsici (Syd.) Butler and Bisby” was carried out in the Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram during 2017- 2023. The study was undertaken with the objective to evaluate P. indica- and its water diffusible exudates- primed chilli seedlings and plants against foliar infection of C. capsici; and study the biochemical and molecular mechanisms involved in this tripartite interaction. A survey conducted in the five agroclimatic zones (ACZs) of Kerala showed that the highest disease incidence (DI) and Percent Disease Index (PDI) were recorded at RARS, Pilicode (DI-90 & PDI-52.60) of northern zone, while the lowest DI was observed at farmer’s field, Kottarakkara (20%) and ORARS, Kayamkulam recorded the lowest PDI (23.63). Chilli anthracnose symptoms namely leaf spot, leaf blight, die-back, fruit rot and mummified fruits were observed in different survey locations of the five agroclimatic zones (ACZ) of Kerala. Nine C. capsici isolates and one isolate of C. gloeosporioides were obtained from the surveyed locations. All the C. capsici isolates produced sparse mycelial growth with concentric zonations of acervuli on potato dextrose agar (PDA) medium. The upper side of culture plates appeared in different shades of white and off-white to grey with regular or irregular margins, while the reverse side looked yellowish brown to black. Isolate Cc4 produced maximum mycelial growth diameter of 8.6 cm compared to 7.2 cm (minimum) in Cc2 on 7th day after inoculation (DAI). Further, microscopic characters such as mycelial width, size of conidia, acervuli, appressoria, number and length of setae were significantly different in the C. capsici isolates. The most virulent isolate of C. capsici was screened based on the lesion size produced on the artificially inoculated leaves and fruits of chilli var. Vellayani Athulya. On 7th DAI, Cc3 isolate of C. capsici produced maximum lesion size of 2.52 cm while isolate Cc4 produced minimum lesions (0.74 cm) on 292 inoculated chilli leaves. Similarly, tender, mature and ripe fruits of var. Vellayani Athulya inoculated with Cc3 isolate produced the highest lesion size (2.48, 2.34 and 2.56 cm respectively) and isolate Cc4 recorded the lowest lesions (1.62, 1.16 and 1.38 cm respectively) on 7th DAI. Thus, isolate Cc3 from Thrissur, was selected as the most virulent isolate of C. capsici. Ten selected KAU/ICAR released varieties of chilli were screened with Cc3 isolate by detached leaf and fruit inoculation method to identify the most susceptible variety. Leaves of Vellayani Athulya recorded the highest lesion size of 2.80 cm on 5 DAI compared to 1.66 cm (lowest) in Manjari. Maximum lesion size of 2.96 cm was observed on the tender fruits of Vellayani Athulya and minimum was recorded on Vellayani Thejus (1.52 cm). Lesions of 3.50 cm (highest) were observed on mature Vellayani Athulya fruits as against 2.00 cm on Manjari fruits. Similarly, ripe fruits of Vellayani Athulya recorded maximum lesion size of 3.46 cm and minimum in Vellayani Samrudhi (1.30 cm) at 7 DAI. Also, in the in vivo study, higher lesion size (3.00 cm) was observed in Vellayani Athulya fruits and lowest (1.62 cm) in fruits of Vellayani Samrudhi at 7th DAI. Standardization of P. indica-colonization in chilli var. Vellayani Athulya was done on plant nutrient medium (PNM) and chilli roots were sampled at different intervals viz., 3rd, 5th, 7th, 10th, and 15th days after colonization (DAC). Young, double-walled chlamydospores were observed within the root cells at 5 DAC. P. indica colonization gradually increased and reached a maximum of 100 per cent at 15 DAC. Dual culture of P. indica and C. capsici showed the appearance of inhibition zone (8.33 DAI) and antibiosis (12.67 DAI) at the point of interaction of the two fungi. Further microscopic observations revealed thickening and lysis of pathogen mycelium by P. indica. The growth of C. capsici was suppressed (3.85 cm) in dual culture compared to normal (6.28 cm) in control at 7th DAI. 57.22 per cent mycelial growth inhibition due to P. indica was observed at 15 DAI. Antagonistic property of P. indica-water diffusible exudates (Pi-WDE) against C. capsici was studied by in vitro poison food technique. Significant 293 differences were observed in the colour and nature of C. capsici growth on PDA media, added with different day-old Pi-WDE in comparison with control. Among them, ten day-old Pi-WDE recorded the highest inhibition (67.30 %) of pathogen mycelial growth whereas the lowest in one day-old Pi-WDE (9.29 %). Mass multiplication of P. indica in the portray medium was carried out. Rapid growth of the endophyte was recorded in a combination of farmyard manure (FYM) and coir pith (1:1), added with 2 per cent gram flour. P. indica completely covered the inoculated trays within 7 DAI. This medium was used to colonize chilli plants with P. indica for further experiments and thus, four treatments viz., P. indica alone (T1), C. capsici alone (T2), P. indica-primed seedlings + C. capsici (T3) and control (T4) were systematized for the in vitro and in vivo evaluation experiments. In vitro study of P. indica-primed chilli seedlings against C. capsici significantly reduced anthracnose lesion size and disease severity to 1.30 cm and 30.63 per cent respectively compared to 2.06 cm and 77.00 per cent in C. capsici alone at 7 DAI. Maximum root and shoot weight were recorded in the P. indicacolonized plants, irrespective of the pathogen. Pot culture experiment was also carried out in the chilli var. Vellayani Athulya. The development of anthracnose symptoms on leaves was delayed by 4 days in P. indica-colonized chilli plants as against 2 days in control. The lesion size and disease severity due to anthracnose was lower in P. indica-colonized chilli leaves (1.58 cm & 54.90 %) compared to C. capsici alone (2.86 cm & 85.20 %) at 10 DAI. Similarly, minimum lesion size and disease severity of 1.17 cm and 27.92 per cent was observed in fruits from pathogen inoculated P. indicacolonized chilli plants than in C. capsici alone (3.56 cm & 91.67 %) at 10 DAI. Field studies were conducted in rabi and summer seasons with two treatments viz., P. indica-colonized and non-colonized plants. P. indica-colonized chilli plants recorded lower anthracnose severity of 27.00 per cent in Rabi and 15.50 per cent in summer compared to 56.50 and 47.00 per cent respectively in control. P. indica colonization improved the biometric characters such as plant height, number of leaves, leaf area, number of secondary and tertiary roots, shoot 294 weight, root weight in primed chilli plants. In addition, yield of chilli was enhanced in endophyte-colonized plants compared to control. Biochemical studies on the mechanism of disease management in P. indica-colonized chilli plants at 0, 12, 24 and 72 hours after inoculation (HAI) showed increased activity of phenylalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (PO). In contrast, the activities of 4-coumaryl CoA ligase (4-CL) and polyphenol oxidase (PPO) were enhanced in chilli plants inoculated with C. capsici alone. Isoenzyme analysis revealed higher induction of isoenzymes of PO and PPO in the P. indica-colonized plants compared to control. Enhanced induction of pathogenesis related (PR) proteins was noticed in the P. indica-colonized plants inoculated with the pathogen at 72 HAI. The capsaicin content was drastically reduced in C. capsici inoculated chilli fruits. However, fruits from P. indica-colonized plants had high capsaicin content (>30% over control) irrespective of infection. Molecular studies of the defense related genes revealed the positive role of P. indica in the management of anthracnose disease in chilli. P. indica reduced anthracnose symptoms in colonized chilli plants by the upregulation of genes involved in phenyl propanoid pathway (PAL1, 4-CL, CAD and CHS), jasmonic acid signaling pathway (PDF1.2 and AOS) and salicylic acid signaling pathway (PR1, EDS1 and PAL3); and downregulating the LOX gene involved in jasmonic acid signaling. In summary, P. indica and its WDE inhibited C. capsici under in vitro conditions. Anthracnose incidence and severity were considerably reduced in the treated chilli seedlings and plants compared to control. In addition, P. indica enhanced growth as well as yield in colonized chilli plants, thereby enhancing the fruit quality. Further, the endophyte improved disease resistance in chilli plants by increasing the defense-related enzyme activities and expression of defense genes. Thus, the root fungal endophyte, P. indica can be considered as an efficient biocontrol agent in the management of chilli anthracnose.Item Evaluation of beneficial fungal root endophyte, Piriformospora indica for the management of Tomato leaf curl virus(Department Of Plant Pathology College Of Agriculture, Vellayani, 2021-12-30) Saru Sara Sam; Joy, MThe research work entitled “Evaluation of beneficial fungal root endophyte, Piriformospora indica for the management of Tomato leaf curl virus” was done at the Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram during the academic year 2019-2021 with the objectives of evaluation of the beneficial fungal root endophyte Piriformospora indica for the management of Tomato leaf curl virus (ToLCV) in tomato; and elucidating the role of reactive oxygen species (ROS) and antioxidant enzymes in the tripartite interaction. ToLCV symptoms were assessed and maintained in insect proof nets from the various field at College of Agriculture, Vellayani, Thiruvananthapuram. The major symptoms observed includes mild curling with yellow speckles, severe leaf curling with blisters on leaf, stunted growth and reduced leaf size with flower abortion. Tomato variety Vellayani Vijay showed a disease incidence of 72.3 to 100 per cent with vulnerability index of 68.5 to 71.0 at different fields, while Anagha variety recorded an incidence of 67.0 per cent and vulnerability index of 54. The presence of ToLCV was confirmed using the universal primers viz., Deng and AV/AC that amplifies the coat protein of Begomovirus which produce amplicons of 520 bp and 575 bp respectively under specified conditions. P. indica-colonization in tomato variety Vellayani Vijay was done in portray mixture amended with 2 per cent gram flour. P. indica spores were observed from 3 day after colonization (DAC) and the spore size increased by subsequent days. P.indica-colonization enhanced the growth parameters including plant height, shoot height, and root and shoot biomass. The tomato plants colonized with P. indica showed an increase in shoot height, number of branches, number of leaves, number of flower, fresh shoot weight, dried shoot weight, fresh root weight and dried shoot weight by 34, 117, 96, 86, 45, 66, 45 and 120 per cent respectively compared to the control at 60 DAC. The days taken for flowering in P. indica-colonized plants were decreased by 12 days and the yield per plant in P. indica-colonized was 892.46 g against 449.51g. P.indica-colonization decreased incidence and severity of fungal and bacterial diseases under field conditions. Moreover, it recorded a drastic reduction in the severity of ToLCD by 58 percent. Tomato plants were subjected to pre- and postcolonization of P. indica challenged with ToLCV through wedge grafting at 2, 5, 10 and 15 days interval. Pre-colonized tomato plants decreased the severity of ToLCD by 66 per cent, while post-colonization decreased the severity by 26 per cent at various intervals of treatments. Further, P. indica-colonization promoted flowering and fruit set against complete flower abortion in non-colonized plants challenged with ToLCV under pot studies. The best treatments selected from pre- and post- inoculation of the fungus include P. indica-colonization followed by graft transmission of the virus after 15 days, and graft transmission of the virus followed by P. indica-colonization after 2 days respectively. Amplification of the virus by polymerase chain reaction (PCR) using Deng and AV/AC primers also revealed that the virus titer was the least in P. indica-pre-colonized plants that were inoculated with the virus after 45 DAC, followed by post-colonized plants that were inoculated with the virus. Nitro blue tetrazolium (NBT) for superoxides and diamino benzidine (DAB) for H2O2 staining recorded a reduction in ROS production in both pre- and post- P. indica-colonized tomato plants artificially inoculated with ToLCV against the virus alone. The decrease in ROS production and disease severity in the fungus colonized plants inoculated with the virus were attributed to the increased total soluble proteins and enhanced activities of antioxidant enzymes. The substantial increase in antioxidant enzymes viz., peroxidase, phosphatase, superoxide dismutase, catalase and glutathione reductase and glutamate synthase were observed in P. indica-colonized plants against the control; and for both pre- and post- P. indica-colonized plants artificially inoculated with ToLCV against the virus alone. Thus, the present study reveals that P. indica can be exploited for the management of ToLCV on prophylactic and curative bases with the best results in the former. P. indica colonized plants enhanced the over all growth and productivity; and drastically reduced the severity of ToLCD under both controlled and open field conditions.Item Analysis of pathotypic variability of Xanthomonas oryzae pv.oryzae, the bacterial blight pathogen of rice and identification of new sources of resistance(Department of Plant Pathology, College of Agriculture, Vellanikkara, 2023-05-11) Aparna, V S.; Raji, PBacterial blight of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is becoming a major production constraint worldwide. In India, the disease is causing considerable yield loss in all rice producing states. In Kerala, the disease is appearing in an epidemic form in recent years in Palakkad, Thrissur and Alappuzha districts. Host plant resistance offers a viable option for the management of the disease. Understanding the pathogen population prevailing in a geographical area is essential for the development of varieties having broad spectrum resistance. The present study was undertaken to analyse the pathotypic variability of Xoo in major rice growing areas of Kerala and to identify new sources of resistance.Item Etiology and management of fungal root rot of cassava in Southern Kerala(Department of Plant Pathology, College of Agriculture , Vellayani, 2023-02-16) Amritha S Kartha; Susha S TharaThe research entitled “Etiology and management of fungal root rot of cassava in Southern Kerala” was conducted in the Department of Plant Pathology, College of Agriculture, Vellayani, during 2020-2022 with an objective of Identification and characterization of major pathogen causing fungal root rot in cassava and its management using different biocontrol agents and fungicides. During this study, root rot infected cassava plants were collected from different locations in the Thiruvananthapuram and Kollam districts of Southern Kerala. Twenty-one isolates of different fungi were obtained from root rot infected parts of cassava. Various symptoms such as yellowing of leaves, rotting of tubers and collar region of cassava stems, mycelial growth over the infected tubers and wilting of cassava plants were noticed in diseased samples from different locations. Based on the pathogenicity test conducted on potted cassava plants of one - month old Vellayani Hraswa variety, T3B (isolate from Thalayal of Thiruvananthapuram district) and K1B (isolate from Ezhukone of Kollam district) isolates were found to cause 100 Percent Disease Incidence (PDI). Based on the number of days taken for disease development, the isolate T3B (Fusarium sp.) was found to be most virulent with a PDI of 100, which took 30 days for symptom development, followed by K1B (Fusarium sp.) with PDI of 100 and 40 days for symptom development. Hence, T3B was selected for further studies. Cultural and morphological characteristics of the T3B isolate were studied. The colony was white cottony, with irregular margins on the front view and a light pinkish tinge on the rear view. Microscopic characters of the T3B isolate revealed that the mycelia were septate and hyaline. Macroconidia and microconidia were produced abundantly on conidiophores. Macroconidia were hyaline, septate (0-2 septa), slender, almost straight and elongated with blunt ends, while the microconidia were slender, hyaline, and single celled with oblong shape. Chlamydospores were absent. Molecular characterization of T3B isolate was carried out using ITS sequencing using universal primers followed by comparative nucleotide sequence alignment with the available database from NCBI. The isolate T3B was confirmed as Fusarium proliferatum with 99.79 per cent homology with F. proliferatum strain OR25F07 (MT476359.1) and the dendrogram was also constructed as a distance tree which showed a phylogenetic relationship. The nucleotide sequence of the T3B isolate was deposited in GenBank and the accession number was retrieved as OQ376433. As part of the study, microflora was isolated from the rhizosphere of healthy cassava plants from Vellayani and Venganoor of Thiruvananthapuram district in Kerala. Among the isolated bacterial and fungal rhizosphere microflora, viz., Aspergillus sp. 1, Aspergillus sp. 2 Bacteria 1 and Bacteria 2, Aspergillus sp. 1 from Vellayani showed the highest inhibitory effect against the pathogen with 64.84 per cent inhibition. The in vitro antagonism of seven biocontrol agents viz., Pseudomonas fluorescens PN026, Bacillus sp. isolate 1 and Bacillus sp. isolate 2 (RRS, Moncompu), Trichoderma sp. 1, Trichoderma sp. 2 (KAU), Piriformospora indica (No. INBA 3202001787) and Aspergillus sp. 1 (selected antagonist) were evaluated against F. proliferatum by dual culture technique and results revealed that highest percentage inhibition was recorded with Trichoderma sp. 1 (57.57 %) followed by Trichoderma sp. 2 (51.51 %). The in vitro evaluation of fungicides through poisoned food technique against F. proliferatum was conducted using eight fungicides viz., mancozeb 75% WP, copper oxychloride 50% WP, propineb 70% WP, carbendazim 50% WP, propiconazole 25 % EC, cymoxanil 8% + mancozeb 64% WP, carbendazim 12% + mancozeb 63% WP and trifloxystrobin 25%+ tebuconazole 50 % 75 WG at 50, 100 and 200 ppm. The highest per cent inhibition was showed by carbendazim 50% WP and trifloxystrobin 25%+ tebuconazole 50 % 75 WG at 200 ppm with an inhibition percentage of 87.87% and 100%, respectively. Carbendazim 50% WP and trifloxystrobin 25%+ tebuconazole 50 % 75 WG were the most effective fungicides used for in vitro assay. A pot culture study was undertaken with the Vellayani Hraswa variety to evaluate the efficacy of effective biocontrol agents and fungicides, viz., Trichoderma sp. 1. Trichoderma sp. 2 (KAU), carbendazim 50% WP and trifloxystrobin 25%+ tebuconazole 50 % 75 WG against F. proliferatum and results shown that the highest disease suppression over control was recorded with Trichoderma sp. 2 (100.00%) followed by Trichoderma sp. 1 (88.89%) and trifloxystrobin 25%+ tebuconazole 50 % 75 WG (77.78%) with sett treatment and soil application at one and two months after planting. The highest yield of 1342.66 g per plant was obtained from treatment with Trichoderma sp. 2 @108 cfu/ml followed by trifloxystrobin 25%+ tebuconazole 50% 75 WG @1 gL-1 (1106.00 g per plant) and Trichoderma sp. 1 @108 cfu/ml (1003.66 g per plant). The present study revealed that Fusarium spp., Lasiodiplodia spp., Colletotrichum spp. and Rhizoctonia spp. were associated with root rot of cassava in Southern Kerala. The most virulent pathogen was identified as Fusarium prolifaratum by analysing the cultural, morphological and molecular characteristics. The sett treatment and soil application of biocontrol agent Trichoderma sp. 2 (KAU isolate) (108 cfu/ml) or the fungicide trifloxystrobin 25%+ tebuconazole 50% 75 WG (1 gL-1 ) at one and two months after planting can effectively manage the disease.