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Central Agricultural University, College of Post Graduate Studies in Agricultural Sciences, Umiam
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ThesisItem Open Access Formulation of bacterial endophyte consortium for the management of Alternaria Blight of mustard(College of Post Graduate Studies in Agricultural Sciences, Central Agricultural University , Imphal, 2018-07) Thokchom, Sushanti; Devi, R. K. TombisanaBroad leaf mustard (Brassica juncea var. rugosa) is one of the most highly consumed rabi vegetable crops of North East India. Alternaria blight, caused byAlternaria brassicae is the major foliar disease infecting the crop at all stages of growth. Bacterial endophytes have been reported to play a major role in the management of plant diseases as well as in plant growth promotion. In the present investigation, fifty six bacterial endophytes belonging to the group Bacillus and fluorescent Pseudomonads were isolated from 20 different cultivars of mustard (Brassica juncea) collected from different regions of North East India. The bacterial endophytes were isolated from seed, root and leaves of these cultivars. Biochemical tests were performed for tentative identification of the isolates upto species level by comparing with the Bergey’s Manual of Determinative Bacteriology. Thirty one isolates of Bacillus were isolated from which 11 were identified as Bacillus cereus, 9 as B. subtilis and 6 as B. megaterium and the remaining 5 as Bacillus sp. Out of 25 isolates of fluorescent Pseudomonad obtained, 13 isolates were identified as Pseudomonas fluorescens, 7 isolates as P. putida, 2 isolates as P. aeruginosa and the remaining 3 as Pseudomonas sp. All the isolates were subjected to dual culture assay against Alternaria brassicae, and from which isolates BS8, BS15, BS21 and FP13 showed maximum inhibition of 59.63, 65.93, 58.52 and 63.33 per cent respectively. When tested for functionalproperties, BS21 was able to produce IAA as well as ammonia, BS15 and FP13 were able to produce only Ammonia whereas BS8 was able to solubilise Phosphate. The formulation (MC) was developed using the 4 efficient compatible endophytes that were screened on the basis of antagonistic and PGP properties. In vitro Plant Growth Promoting activity showed Vigour Index (VI) of 714.55 in the seeds of cultivar MKH2 treated with MC (108 cfu/ml) as compared to the Control which gave VI of 412.5. In vivo experiment was conducted in which the treatment consisting of seed+ seedling root dip+ foliar application with MC showed Percent Disease Index (PDI) of 16.30±0.74 compared to positive control which gave PDI of 52.59±3.92. The formulation may be further tested for its efficacy in field condition. Studies on mass production of the formulation with efficient carrier system may be taken up in order to provide a safer and efficient management of Alternaria Blight of Mustard.ThesisItem Open Access Evaluation of potential bacterial endophytes against major vegetables pathogens(College of Post Graduate Studies in Agricultural Sciences, Central Agricultural University , Imphal, 2018-07) Malakar, Pranab; Majumder, DipaliUtilisation of the endophytes against plant pathogen is considerd as successful means for plant disease management. Bacterial endophytes have beneficial effects on host plants in terms of growth promotion and in suppression of diseases. Fungicides used for plant disease management leads to the residue deposition and known to cause various environmental and health hazards. Native endophytes could be the alternative for chemicals as a component of integrated disease management. Keeping in mind, the present investigation has been formulated to evaluate the potential bacterial endophytic antagonists against major vegetable pathogens. Forty two (42) bacterial endophytic isolates have been evaluated against four major vegetable pathogens viz., Colletotrichum lindemuthianum, Phomopsis vexans, Alternaria brassicicola and C. capsici. Although, most of the bacterial endophytes showed inhibition against tested fungal pathogens. However, NGB 21 showed best inhibition against C. lindemuthianum (60.49 %) and C. capsici (55.92 %), isolate BE 1 against P. vexans (71.85 %) and SVC 11 was recorded best inhibition against A. brassicicola (63.70 %). Four screened bacteria viz., NGB21, NE 1, SVC11 and MIW1 stresses were evaluated for functional properties and abiotic. NGB21was found to have better functional property (More production of ammonia and solubilising insoluble phosphate) and can grow in different abiotic stress conditions (low pH upto 4.5 and higher aluminium concentration of 150 and 200 ppm). Screened isolates were also evaluated for plant growth promotion and disease suppression on French bean (anthracnose disease) in the field condition. The bacteria were treated alone and in consortium mode along with different combinations of treatments. Seed treatment with microbial consortia (STMC), soil application of MC refore sowing (SMCBS) and fungicidal spray (carbendazim) recorded for less disease incidence (14.44 % at 81 DAS) while STMC recorded for maximum disease incidence (32.77 % at 81 DAS) among microrial consortia combinations evaluated. PGP attributes of MC were also evaluated where it was observed that showed STMC + SMCBS + FS better performance compared to the other treatments. Isolates NGB21 and BE 1 as well as the method of application i.e., STMC + SMCBS + FS could be further tested for their antagonism and plant growth promotion under field condition in Meghalaya for further use as a component of integrated disease management for vegetable cultivation.ThesisItem Open Access Evaluation of bacterial endophytes against ginger rhizome rot(College of Post Graduate Studies in Agricultural Sciences, Central Agricultural University , Imphal, 2018-08) Bamon, Meshanki; Majumder, DipaliGinger (Zingiber officinale Rosc.) is a major spice crop in India and Meghalaya is the third largest producer in the country. Rhizome rot, commonly known as soft rot is a major constraint in ginger cultivation. With the increase in awareness about the hazardous effects of the chemicals, exploitation of effective microorganisms like endophytes has been increasing witha greater pace for management of plant diseases. Keeping that in mind, the present investigation was formulated to evaluate 6 potential bacterial endophytes against pathogens associated with ginger rhizome rot complex in Meghalaya. Six potential bacterial ginger endophytes (GE-1, GE-2, GE-3, GE-4, GE-5 and GE-6) were screened based on several functional properties. It was recorded for that all the tested bacterial antagonists do not produced HCN, whereas all isolates were positive for ACC deaminase. Highest halo production for siderophore was recorded for GE-1 (21.67mm). Five isolates produced chitinase and protease enzymes except GE-2, whereas 3 isolates were recorded for production of cellulase (GE-1, GE-2 and GE-6) and amylase (GE-2, GE-3 and GE-6). None of the isolates produced pectinase enzyme. Four isolates were found positive for IAA production, whereas only one isolate GE-5 recorded for ammonia production. Phosphate (P) and Zinc (Zn) solubilisation test revealed that only GE-5 (1.17mm) was positive for P solubilisation, whereas GE-1 (0.29 cm2) and GE-5 (0.01 cm2) solubilised Zn. Growth of all isolates was observed in the pH ranged from 4 to 7. Alluminium (Al) tolerance tested revealed that only isolate GE-2 showed very good growth at Al concentration of 50 and 100 ppm, followed by GE-1, showed fairly good and very good growth at 50 and 100 ppm respectively. All isolates were found compatible with 5 fungicides tested viz., Copper oxychloride, Carbendazim, Mancozeb, Metalaxyl and Bordeaux mixture in all concentrations (0.05%, 0.1%, 0.2% and 0.3%).The bacterial endophyte GE-1 and GE-3 were insensitive to tetracycline and streptocycline at all concentration whereas, the other isolates were sensitive. Dual culture assay of isolates against pathogens associated with ginger rhizome rot complex viz., Pythium myriotylum, Fusarium sp, Ralstonia solanacearum revealed that the isolate GE-1 showed 80.98% suppression of P.myriotylum, GE-6 (67.85%) against Fusarium sp. and GE-3 (78.89%) against Ralstonia solanacearum. These endophytes were further evaluated growth promotion and disease suppression. In the field experiment, GE-1 was recorded with the greatest sprouting (98.89%), lower soft rot incidence (19.83%) and higher yield (0.59 kg/ plot) compared to chemicals like Mancozeb (90.0%, 12.58.0% and 0.2 kg/plot, respectively) and positive control (65.5%, 40% and0.13 kg/ plot, respectively). Based on the performance of endophytes, microbial consortia was prepared by using 3 better performed endophytes (GE-1, GE-3 and GE-6) and tested against pathogens of rhizome rot complex during storage in pits. Disease reduction for the rhizomes treated with microbial consortia under storage condition was recorded as 25%. GE 1 showed the highest population (1.6 x 1011 cfu/ml) in Trehalose (5 mM) amended medium, GE3 in Trehalose (15mM) amended medium and GE6 in glycerol (10 mM) amended medium for shelf life experiment. The experiment also revealed that all the isolates were found positive for siderophore production and negative for HCN production during storage (upto 60 days). It could be concluded from the present investigation that the bacterial endophytes viz,.GE-1, GE-3 andGE-6 could be successfully used for development of microbial consortia against ginger rhizome rot complex upon further evaluation under different field conditions of Meghalaya. Application of liquid bioformulation in consortia mode, amended with trehalose (15 mM) of these effective endophytes could be an important component of integrated management of ginger rhizome rot complex in Meghalaya.ThesisItem Open Access Variability studies of Phomopsis vexans (Sacc. & Syd.) Harter and eco-friendly management of fruit rot of Brinjal(College of Post Graduate Studies in Agricultural Sciences, Central Agricultural University , Imphal, 2018-08) Jamir, Moakala; T. RajeshBrinjal (Solanum melongena L.) also known as aubergine or eggplant, a member of solanaceae family is a popular vegetable crop grown almost worldwide.Several diseases invading the crop are the major constraints for limited brinjal production and productivity. The most destructive and important disease on brinjal is leaf blight and fruit rot incited by the fungus, Phomopsis vexans (Sacc. & Syd.) Harter. The fruit rot phase adversely affects the fruit quality, quantity and ultimately reduce the market value. The repeated isolation from infected brinjal fruits and leaf blight symptoms revealed the association of Phomopsis spp, which was identified after the cultural and morphological studies as Phomopsis vexans. A total of 20 isolates of P.vexans were obtained from Ri-Bhoi district of Meghalaya. Though all the isolates were found pathogenic, isolate Pv6 was found to be highly pathogenic. The isolates showed significant variations in colony colour, shape, zonation and consistency in the media viz., Potato dextrose agar (PDA), Malt extract agar (MEA), Richard’s synthetic agar (RSA) and Czapek’s dox agar (CDA). The maximum growth of all the isolates was supported by PDA. The highest biomass production was recorded in isolate Pv21 (393mg) and the lowest in isolate Pv12 (163 mg). The isolates also produced spherical, black pycnidia which were small or big except isolate Pv14 which produced minute size and abundant. Alpha (α) and beta (β) conidia were also observed in all the isolates.The α conidia size was maximum in isolate Pv5 (8.9x3.4μm) and minimum in isolate Pv15 (4.9x2.8μm), while the β conidia size was maximum in isolate Pv17 (23.56x2.26μm) and minimum in isolate Pv4 (14.88x1.27). The physiological conditions such as pH 7 and temperature 27°C were found to be most suitable for mycelial growth of all the isolates. Under in vitro condition garlic extract (15% w/v) showed the maximum mean inhibition of mycelial growth (100%) while ginger extract (5% w/v) gave the lowest mean inhibition of mycelial growth (77.29%). Dual culture assay carried out with the bio-control agents showed that Trichoderma harzianum recorded the highest mean inhibition (77.76%), whereas Pseudomonas fluorescens displayed the lowest mean inhibition (39.78%) against the five isolates tested. Further, in vitro detached fruit technique results revealed that garlic extract (10% w/v) was found effective against fruit rot followed by T. harzianum in the test isolate Pv6.ThesisItem Open Access Management of Alternaria spp. causing Black leaf spot of cauliflower in Meghalaya(College of Post Graduate Studies in Agricultural Sciences, Central Agricultural University , Imphal, 2018-07) Papang, Heipormi; Devi, R. K. TombisanaCauliflower suffers heavily from black leaf spot disease and found well distributed in Meghalaya. PDI was found maximum in Ri Bhoi district (52.46%) followed by West Jaintia Hills (44.75%). In Ri Bhoi District, maximum PDI was found in Umroi Pyllun (62.03%) whereas in West Jaintia Hills, maximum PDI was found in Jowai (60.18%). A. brassicicola was consistently associated with black leaf spot of cauliflower. Maximum growth was observed in Corn meal agar but maximum sporulation was obtained from Potato dextrose agar. Growth and sporulation for A.brassicicola was found optimum at temperature range of 25 to 30ºC and pH 6 to 6.5.Light conditions revealed that continuous light gave maximum growth but maximum sporulation obtained from alternate light conditions of 12 hours light/12 hours dark. Extract from pitcher plant at 10% and 15% showed 100% inhibition, Garlic at 15%(83.78%), pitcher plant at 5% (82.87%), winged prickly ash at 15%(82.59%) were the most effective in inhibiting the growth of the pathogen. Phytochemical analysis found that all botanicals contain at least one phytochemical with maximum was found in winked prickly ash and ginger among all the phytochemicals tested. Trichoderma harzianum showed maximum inhibition (81.11%) followed by T. viride and B. Subtilis (CoB5Y1) with 62.41% and 52.96% inhibition respectively whereas B. subtilis (BC07) showed the least inhibition by 28.34%. Mancozeb gave maximum inhibition at 0.1%,0.2% and 0.25% concentrations by 84.64%, 96.11% and 100% respectively and least inhibition was found on azoxystrobin at 0.1% concentration with 39.72% inhibition.Winked prickly ash was most compatible with T. harzianum as it gave least inhibition at 5% whereas T. viride found to be most compatible with derek at 5%. Among the fungicides, T. harzianum and T. viride showed less inhibitory effect at 0.1% copper hydroxide and azoxystrobin respectively. Among bacterial antagonists, all were found compatible and combination of two strains of B. subtilis (BS217+CoB5Y1) was the most effective with 75.93% inhibition of growth. Field evaluations showed that the treatment of T. harzianum combined with copper hydroxide was significantly superior with less per cent disease index (16.61) and highest yield (42.27t/ha).ThesisItem Open Access Studies on Asochyta phaseolorum sacc. causing Ascochyta blight on cowpea and its management(College of Post Graduate Studies in Agricultural Sciences, Central Agricultural University , Imphal, 2018-08) B. K. Namriboi; Devi, R. K. TombisanaDuring survey, cowpea [Vigna anguiculata (L) Walp.] was found severely affected by ascochyta blight and the disease was found well distributed in the surveyed areas of Meghalaya. The per cent disease incidence was found maximum in Mawlai (52.34%) under East Khasi Hills followed by Umran (45.57 %) under Ri-Bhoi district. On average, PDI was found highest in Ri-Bhoi district (37.50%) and least in West Khasi Hills district (15.47%). Ascochyta phaseolorum was consistently isolated from symptomatic leaves. A total of nine isolates of A. phaseolorum were isolated from the surveyed areas. The isolates showed variations in colony diameter, colour, shape, zonation, appearance and sporulation. Potato Dextrose Agar (PDA) was found favourable for the growth of all nine isolates whereas minimum average growth was in Malt Extract Agar (MEA). PDA and Oat Meal Agar (OMA) supported maximum sporulation of all the isolates. The size of pycnidia were 73.7-112.2×55.6-87.2μm and the average pycnidiospores size varied from 12.1-14.5×2.9-5.3μm. Mycelial growth in dark and the light conditions varied where alternate cycle of 12 hrs light and 12 hrs darkness supported maximum growth and sporulation. Growth and sporulation was found optimum at pH 6 and pH 6.5 for all the isolates whereas pH 4.5 supported least growth. The optimum temperature for mycelial growth and sporulation was found at 25°C while at 4°C no growth was recorded for all the isolates. Plant extracts, biological control agents and chemical fungicides were evaluated in vitro against the growth of the pathogen. Garlic extract at 5% showed the highest per cent inhibition (86.91 ± 1.07%) of mycelial growth followed by turkey berry at 15% (82.33 ± 0.44%). T. harzianum gave the highest overall per cent inhibition (65.93 ± 0.37%) against the pathogen followed by T. viride (60.86 ± 0.24%) and P. fluorescens could inhibit 25.56 ± 0.47%. Propiconazole, thiophanate-methyl and carbendazim at 0.05, 0.1 and 0.15 and mancozeb at 0.2 and 0.25 per cent concentrations gave 100% inhibition of mycelial growth of the pathogen. These could be further evaluated under in vivo condition to confirm their efficacy and could be included as components for integrated disease management for ascochyta blight of cowpea.ThesisItem Embargo Study on phoretic and mycangial fungi of Ambrosia Beetles and their aggressiveness associated with Tree Bean decline.(College of Post Graduate Studies in Agricultural Sciences, Central Agricultural University - Imphal, 2022-11) Angom, Langlentombi; Singh, Akoijam RatankumarParkia timoriana, popularly known as tree bean is an underutilised yet highly potential crop in north eastern region of India. Since, last two decades, widespread occurrence of sudden death and decline of matured tree bean were reported from Manipur, Mizoram, Meghalaya, India. It has been reported that the tree bean decline disease is caused by Fusarium spp. mainly (F. euwallaceae, F. ambrosium, F. solani) fungal symbionts of ambrosia beetles, resulted significant reduction in pods production. Ambrosia beetles belongs to weevil subfamilies; Scolytinae and Platypodinae and survive in symbiosis with fungal symbionts. Ambrosia beetles carries their nutritional mutualistic fungi in specialized structures called mycangia. Fungal propagules are also dispersed phoretically on the beetle’s exoskeleton. Phoretic transmission of symbiotic fungi, Fusarium euwallaceae of the Euwallacea sp. of ambrosia beetles potentially contribute to the infection in tree bean. In this research, six different species of ambrosia beetles infesting the tree bean decline were identified using morphology and molecular, viz; Euwallacea interjectus, Euwallacea similis, Euwallacea fornicatus, Euplatypus parallellus, Xylosandrus crassiusculu and Xyleborinus sp. Identification was carried out by amplifying the mitochondrial gene COI region, Cytochrome Oxidase I, using the primer pair LCO1490 and HCO2198. Fungal isolation from each species (mycangia and phoretic) of the ambrosia beetle was done by serial dilution, to determine the abundance of symbionts and other species. F. euwallaceae recorded the highest frequency in mycangia and recovered from the beetles, E. interjectus, E. similis and E. Fornicates followed by different species such as Fusarium ambrosium, Fusarium solani. Other fungi, Nectria pseudotricha, Clonostachys rosea Lasiodiplodia theobromae, and L. pseudotheobromae that was also recovered from external isolation of fungi from the beetles. Average CFUs per beetle ranged from 14.55 to 32.05 and CFU ranged from 1 to 112. The highest CFUs (34.4/beetle) in F. euwallaceae recovered from Euwallacea interjectus, followed by E. fornicates (33.55/beetle) in phoretic whereas in internal, E. similis recorded the highest (32.05/beetle). Comparing between internal and external isolation of fungi, it was observed that frequency of recovery of fungi from external isolation was found greater than mycangial fungi. In another experiment, pathogenicity test was assayed on 2 years old tree bean seedlings to check the aggressiveness using four fungal isolates viz. Fusarium solani, Lasiodiplodia theobromae F. euwallaceae, and F. ambrosium under net house condition. Agar plug (5mm) and spore suspension technique (2×106 conidia/ml) was used to check the aggressiveness of the selected pathogens. Lesion was developed in all treatments except the control with the highest lesion length of 10.48±1.08 cm using agar plug and in spore suspension, the highest lesion length recorded was 6.88±0.57cm. All the isolates showed statistically similar aggressiveness to the host. In stem detached method, the highest lesion length recorded was 7.04±0.28 cm. In this present study, it can be concluded that different ambrosia beetles infesting tree bean and carry their symbiont fungi, especially F. euwallaceae and symbionts were found pathogenic. In future, further studies may be conducted on diversity of ambrosia beetles, symbiotic fungi and their host ranges and susceptibility in this region and at instance, identification of resistance tree beans germplasms against this pathogens and pest may also be explored.ThesisItem Open Access Standardization of shiitake mushroom (Lentinula edodes (Berk).) pegler) production technology in Meghalaya / by Nandeesha S V(College of Post Graduate Studies in Agricultural Sciences, Central Agricultural University, Imphal, 2019) S. V., Nandeesha; Rajesh, T.Shiitake mushroom Lentinula edodes (Berk.) Pegler, is one of the six popular edible mushrooms and it is a wood rotting fungi capable of decomposing the cellulose and lignin structural components which is having several medicinal properties. The present study was undertaken to evaluate different locally available grain substrates viz., paddy, maize, pearl millet, sorghum, wheat, saw dust and wood pieces of jack fruit, willow and pine tree for spawn production by using three strains, DMR 388, DMR 32 and Meghalaya Local. The results revealed that maximum mycelial growth on PDA media was observed in the strain, Meghalaya Local (89.74 mm) at 20th day after inoculation whereas the minimum mycelial growth was seen in DMR 388 (85.40 mm). Among three strains and 12 different substrates used for spawn production, the fastest growth of mycelium was observed in sorghum grains (14.40 days) for strain Meghalaya Local whereas the highest number of days taken by saw dust of pine (60.00 days) for strain DMR 388 compared to other strain. In another experiment, different agricultural wastes such as wheat straw, paddy straw, maize stubbles, maize shelled cobs, rice bran and saw dust of jack fruit were tested as substrates for cultivation of shiitake mushroom by using three strains, DMR 388, DMR 32 and Meghalaya Local. Rapid mycelial growth was found in wheat straw substrate for DMR 388 (40 days). But, mycelial growth was very much delayed in paddy straw substrate for strain Meghalaya Local (62 days). Wheat straw substrate showed the least number of days for mycelial coat formation (48.4 days), bump formation (61.0 days), browning (71.2 days), fruiting (74.2 days) and harvesting (76.0 days) for strain DMR 388. The highest number of days was taken for mycelial coat formation (72.8 days), bump formation (94.6 days), browning (112.4 days), fruiting (123.6 days) and harvesting (129.6 days) in paddy straw substrate for strain Meghalaya Local. The growth parameters such as the highest stalk length (56.6 mm), stalk diameter (15 mm), pileus diameter (69.6 mm) and pileus thickness (16.2 mm) were observed in wheat straw substrate for DMR 388. Lowest stalk length (42.6 mm), stalk diameter (9.4 mm), pileus diameter (51 mm) and pileus thickness (11 mm) were seen in paddy straw substrate for strain Meghalaya Local. The yield attributes showed that average number of fruiting bodies per bag and average fresh weight of mushrooms were found highest in wheat straw substrate (14.4 and 300 g) for strain DMR 388. But, lowest average number of fruiting bodies per bag and average fresh weight of mushrooms were found on paddy straw substrate (4 and 194 g) for strain Meghalaya Local. The highest biological efficiency were recorded in wheat straw substrate (37.50%) for strain DMR 388 and the lowest biological efficiency was seen in paddy straw substrate (24.25%) for strain Meghalaya Local.ThesisItem Open Access Cultivation of ganoderma lucidum (W.Curt.:Fr.) P. karst and its antifungal properties against phytopathogenic fungi(College of Post Graduate Studies in Agricultural Sciences, Central Agricultural University, Imphal, 2019) Roopesh, M.; Devi, R. K. TombisanaIn the present study G. lucidum (W.Curt.Fr.) P. Karst was collected from wild. The mushroom was isolated by tissue culture and purified by hyphal tip cut method. Microscopic studies revealed that the mycelia produced clamp connections, fusiform chlamydospores and basidiospores were brown, ovoid and double walled with inter wall pillars separating the two layers. Spawn production on wheat grains took 15 days for complete mycelial colonization. The cultivation studies on different substrates, earliest duration (21 days) for spawn run on PS1 as compared to other substrates. Earliest fruiting primordia initiation (23 days) was observed on PS1, whereas 24 and 28 days on WS2 and SB3. No primordia formation was observed on sheesham sawdust at all. Earliest fruiting maturity (16 days) after primordial initiation was observed on SB3 even though it took long duration for primordia initiation, however a maximum duration of 20 days was recorded on WS3. A maximum stipe length of 5.37cm and with a pileus diameter of 4.77 cm were recorded on PS1. Biological efficiency was found highest (11.43%) on PS1 followed by WS2 (10.7%) and SB3 (9.18%) respectively. Maximum biological efficiency of G. lucidum (11.43%) was obtained from PS1 but lowest from PS3 (6.41%). Phytochemical analysis revealed the presence of carbohydrates and steroids but absence of alkaloids and flavonoids. Maximum antifungal activity of phytochemicals from aqueous (10.93 mm to 16.93 mm), methanol (11.90mm to18.66 mm) and acetone extracts (14.06 to 20.67 mm) against Aspergillus niger at 5, 10 and 15 % concentrations, whereas minimum against Sclerotium rolfsii (9.25 mm to 14.80 mm).
