Browsing by Author "Reshmy Vijayaraghavan"

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Alginate based encapsulation of pseudomonas fluorescens for management of soil borne pathogens
(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2020) Sivadharshnapriya, R; Reshmy Vijayaraghavan
Biological control, an eco-friendly and cost effective approach for plant disease management in agriculture has been practiced for several decades. Pseudomonas fluorescens, one such biocontrol agent is used to combat many phytopathogens. For commercial use, microbial inoculum should be supported by an appropriate formulation preventing a rapid decline of introduced microorganisms and extending their shelf-life. Various formulations available in the market are powder, liquid and granular formulations where such carrier based inoculants which generally faces problems like poor shelf life, high chances of contamination, bulk sterilization problem, unpredictable field performance and sometimes unavailability of good carrier materials. In order to overcome the disadvantages of these formulations, microencapsulation is one such alternate viable option prepared by using sodium alginate as a polymer which results in extended shelf-life and controlled microbial release from the formulation thus, enhancing their application efficacy. Hence, the present study was undertaken in the Department of Plant Pathology, College of Horticulture, Vellanikkara to develop alginate based formulation of Pseudomonas fluorescens for the management of soil borne pathogens. P. fluorescens, the reference culture of KAU was used for preparation of alginate beads. To improve the shelf life of alginate beads, King’s B broth was enriched with adjuvants viz., sugar source (mannitol and trehalose), wetting agent (PVP and PEG), adhesive (CMC and liquid paraffin) and surfactant (tween-80) in nine different treatments and was evaluated at monthly intervals. After nine months of observation, maximum population of P. fluorescens was recorded in treatments T1 (mannitol +PVP +CMC +tween-80) (1.33 x 108 cfu ml-1) and T3 (mannitol +PEG +CMC +tween-80) (1.66 x 108 cfu ml-1) compared to control which were selected for the preparation of beads. The beads were prepared from the above selected treatments as per the protocol of Bashan et al. (2002) with modifications. The beads were prepared in three different batches viz., beads from alginate alone, beads from alginate amended with skim milk and beads after secondary multiplication. Various parameters were standardised in order to prepare effective bead formulation. Beads from both alginate and alginate amended skim milk formulations produced from higher concentration of sodium alginate (3%) and calcium chloride (3.5% and 3.0%) solution with 60 min of curing time and 9 to 15 cm of falling distance produced perfectly spherical beads with maximum diameter of above 1.70 mm, higher bead weight of above 16.6 mg with more than 60 per cent bead yield. Such beads showed reduced swelling percentage which holds higher per cent of water content inside beads and lowest shrinkage percentage that facilitates higher survival and slow release of the bioagent for a longer period of time. Shelf life of P. fluorescens encapsulated in alginate beads alone prepared from two best treatments T1 (mannitol + PVP + CMC + tween-80) and T3 (mannitol + PEG + CMC + tween-80) showed a higher shelf life compared to alginate amended skim milk beads and beads after secondary multiplication. Higher bacterial entrapment were observed in alginate beads prepared from sodium alginate (3%) and calcium chloride (3.5% and 3.0%) respectively in treatments T3A12 (10.33 x 1020 cfu g-1) followed by treatments T3A11 (8.33 x 1020 cfu g-1), T1A6 (6.33 x 1020 cfu g-1) and T1A5 (5 x 1020 cfu g-1) respectively after four months of preparation. The alginate bead combinations B-1: T1 (mannitol + PVP + CMC + tween - 80) + sodium alginate (3%) + CaCl2 (3%) and B-2: T3 (mannitol + PEG + CMC + tween -80) + sodium alginate (3%) + CaCl2 (3%) were selected for in vitro evaluation studies against major soil borne pathogens and it was noticed that these formulations inhibited soil borne pathogens viz., Pythium aphanidermatum (100%) followed by Phytophthora nicotiana (72.22 and 77.77%) Ralstonia solanacearum (70.36 and 74.07%) and to some extent inhibition of Fusarium oxysporum (27.77 and 30.55%). However, no inhibition was observed on the growth of Sclerotium rolfsii and Rhizoctonia solani under in vitro conditions. Hence the study has clear by demonstrate a protocol to produce microbeads of P. fluorescens which are less bulky, non-toxic, biodegradable and enables slow and controlled release of the biocontrol agent and thus could maintain a bacterial population for a relatively longer period.
Cataloguing and documentation of fungal diseases of gerbera
(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2016) Praveen, N M; Reshmy Vijayaraghavan
Cataloguing, documentation and management of fungal diseases of strawberry (Fragaria x ananassa Duch.)
(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2017) Amrutha, P; Reshmy Vijayaraghavan
Characterization and evaluation of Pseudomonas spp. for abiotic stress tolerance
(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2019) Reshma, K S; Reshmy Vijayaraghavan
Exploitation of abiotic stress tolerant strains of trichoderma spp. for the management of soil borne fungal pathogens
(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2018) Stella Doncy, P P; Reshmy Vijayaraghavan
Soil borne phytopathogenic fungi are known to cause severe yield loss of several crops. To mitigate these crop ailments, farmers mostly rely on chemical methods of disease control such as fungicides and other pesticides which are deleterious to the environment. To date, biological control is an ecofriendly approach for the effective management of crop diseases. The fungi belonging to the genus Trichoderma are one among the most exploited biocontrol agents in the field of agriculture. However, the performance of Trichoderma spp. gets reduced when it is exposed to abiotic stressed conditions such as drought, high temperature, salinity, acidity and fungicides. Hence, this study was proposed to identify and exploit stress tolerant isolates of Trichoderma spp. with antagonistic potential in Kerala. Intensive soil sampling surveys were conducted across different stressed ecosystems of Kerala viz., Palakkad, Alappuzha, Vytilla, Kumarakom, Wayanad and Thrissur for the isolation and enumeration of native Trichoderma spp. A total of 24 isolates were obtained from 52 soil samples collected from different locations. Based on the number of Trichoderma spp. obtained from each district, they were serially numbered and abbreviated according to the name of the location. Accordingly, PAT 1 to PAT 6 represents number of isolates of Trichoderma spp. from Palakkad district, ALT 1 to ALT 3 from Alappuzha, VYT 1 and VYT 2 from Ernakulam district, KUT 1 from Kottayam district, WAT 1 to WAT 8 from Wayanad and THT 1 to THT 4 from Thrissur district. Cultural and morphological identification of these isolates were carried out under in vitro conditions. Isolates of Trichoderma spp. were subjected to in vitro screening for abiotic stress tolerance such as high temperature, drought, acidity, salinity and also to test their sensitivity towards copper fungicides. The isolates PAT6 and WAT2 were found as thermotolerant, VYT2 and ALT 1 as drought tolerant, ALT 3 and ALT 1 as acid tolerant and saline tolerant and the isolates ALT1, ALT3 and PAT 1 as copper fungicide tolerant. The selected six isolates were further subjected to biochemical tests and the study showed that the isolates VYT 2, ALT 3 and ALT 1 showed highest cellulase, β- 1, 3 glucanase and protease activity. Likewise, isolates PAT 1, ALT 1 and ALT 3 were found as best producers of ACC deaminase and PAT 1 and ALT 1 as the best cytokinin producer. The best performing isolates (ALT 1, ALT 3, PAT 1, PAT 6 and VYT 2) after enzyme study were subjected to dual culture experiment with five major soil borne pathogens to test their antagonistic potential. The isolates ALT 3, ALT 1, PAT 6 and PAT 1 showed more than 70 per cent inhibition of R. solani whereas, isolates ALT1 and VYT 2 showed only 57.78 and 55 per cent inhibition of S. rolfsii respectively. However, no significant difference was noticed among the isolates when grown against F. solani. Cent per cent inhibition of P. aphanidermatum was noticed with Trichoderma isolates PAT 1 and ALT 1. All five isolates showed 100 per cent inhibition on the growth of pathogen, P. capsici. Among the five, four isolates viz., ALT 1, ALT 3, PAT 1 and PAT 6 with best antagonistic potential were subjected to molecular characterization at Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram and the isolates showed homology with the nucleotide sequence of Trichoderma asperellum. A pot culture experiment was laid with the selected isolates viz., ALT 1, ALT 3, PAT 1 and PAT 6 to test the growth promotion of cowpea and biocontrol efficacy against Rhizoctonia solani. It was observed that the isolate PAT 6 coated seeds showed 100 per cent germination and also recorded better biometric characters and yield. Moreover, the lowest per cent disease incidence of 11.11 per cent was only recorded with both the isolates ALT 3 and PAT 6. Thus, the study has enlightened our knowledge on the existence of abiotic stress tolerant isolates of T. asperellum which can be employed in future for the biocontrol of soil borne pathogens in such conditions.
Management of Phytophthora disease in black pepper nursery
(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2003) Reshmy Vijayaraghavan; Koshy Abraham
Phytophthora rot is the serious disease of black pepper nursery. An investigation was carried out to isolate and select the efficient antagonists from black pepper nurseries and use them alone or in combination with fungicides in the integrated management of the disease. The experiment was laid out at CCRP farm at College of Horticulture, Vellanikkara, The pathogen causing the disease was isolated and identified as Phytophthora capslcl Leonian emend A. Alizadeh and P.H.Tsao based on the cultural and morphological characters. Quantitative estimation of rhizosphere microflora from different pepper nurseries yielded more soil bacteria followed by fungi and actinornycetes. All the 22 fungi, five out of 20 bacteria and none of the actinomycetes tested were antagonistic to P. capsici. Among the fungal isolates, 13 isolates including standard culture of T. harzianum recorded cent per cent inhibition of P. capsici. Further, selection of the efficient isolates was carried out based on the antagonistic index (AI). The isolates 22 F and 34 F recorded an AI of3000 and 1500 respectively and these were identified as Trichoderma longibrachiatum and Trichoderma viride. The standard culture of T harzianum also recorded an AI of 1500. The three antagonists were found parasitic on Picapsici as evidenced by excessive coiling, penetration and disintegration of the hyphae. The fungicides viz., Bordeaux mixture, Kocide, Captaf and Kavach were incompatible with the three antagonists, while, lndofil M-45, Ridomil I\.1Z, Akomin and Anthracol were compatible. Fytolan showed partial compatibility with Tviride and 1~ harzianum but incompatible with T. longibrachiatum. Among the eight insecticides tested, Phorate and Carbofuran showed compatibility with the antagonists, whereas Monocrotophos, Quinalphos, Endosulfan, Dimethoate, Cypennethrin and higher concentration of Chlorpyrlphos were incompatible. In general, fertilizers like Urea, Rajphos, Ammonium sulphate and Muriate of potash (MoP) were compatible with antagonists, while, Factomphos and higher concentration of Urea did not support good growth. 149 Bordeaux mixture, Fytolan, Kocide, Indofil M-45, Ridomil MZ and Captafat all concentrations and higher concentration of Akomin-40 and Anthracol were inhibitory to P. capsici. The insecticides Phorate, Carbofuran and Chlorpyriphos showed comparatively good inhibitory effect against the pathogen but complete inhibition of pathogen was noticed with Monocrotophos, Endosulfan, Quinalphos, Dimethoate and Cypermethrin. The fertilizers viz., urea, MoP, Rajphos supported growth of the pathogen while, Factomphos and ammonium sulphate exerted an inhibitory effect. Solarization of potting mixture resulted in the build up of soil temperature and the build up was more in the upper layer of soil. Solarization of potting mixture and application of biocontrol agents had a positive effect in increasing the sprouting and reducing the pre-sprouting mortality of cuttings and is comparable to plants raised as per PoP. Observations on the incidence and severity of Phytophthora rot in black pepper showed that in general soil solarization, application of antagonists and spraying of Ridomil MZ had a favourable effect in checking the disease and the effect is almost similar to that of disease management as per PoP. A variation in the population of soil microfIora in different treatments was o~~rved. Thy cuttings raised in solarized potting mixture incorporated with native antagonists had a significant effect in increasing the height and number of leaves.
Microencapsulation of Trichoderma viride for management of major soil borne fungal pathogens
(Department of Plant Physiology, College of Agriculture, Vellanikkara, 2020) Saleena, M; Reshmy Vijayaraghavan
Plant growth promoting rhizobacteria mediated induced systemic resistance against bacterial wilt in ginger
(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2007) Reshmy Vijayaraghavan; Koshy Abraham
The pathogen causing bacterial wilt of ginger was isolated and identified as Ralstonia solanacearum biovar III based on its cultural, morphological and biochemical characters coupled with pathogenicity. Rhizosphere microflora of ginger from different locations of Thrissur, Waynad and Palakkad districts were isolated. Altogether, 163 rhizobacterial isolates were selected from these areas and their antagonistic activity against the pathogen assessed. Out of 163 isolates, only 45 showed antagonistic reaction. Further, study of these antagonists based on zone of inhibition resulted in selection of 20 isolates. The effect of these 20 isolates in promoting the growth of ginger was studied in pot culture in comparison with three reference cultures of P. fluorescens and B. subtilis. Result of this experiment revealed that only 11 isolates including the two reference cultures of P. fluorescens had growth promoting effect as evidenced in terms of yield and yield attributing characters of ginger. Factors which impart growth promotion in ginger by these isolates were assessed by estimating the inhibition zone, vigour index, hydrogen cyanide, indole acetic acid, ammonia production and ‘P’ solubilzation and based on that, PGPR index of the isolates was worked out. In addition to that, production of salicylic acid, antibiotics and siderphore by the isolates, the secondary metabolites which are known to play a role in disease suppression were assessed. The isolates varied in their ability to produce salicylic acid. Isolates RB-22 followed by RB-11, RB-144 and RB-66 produced more number of antibiotics which include pyoluteorin, pyrrolnitrin, 2,4DAPG etc. Similarly, isolate RB-22 and RB-11 produced maximum siderophores. The potential of these 11 rhizobacterial isolates in imparting resistance against the disease was assessed in another pot culture experiment by estimating phenol, proteins and amino acid content of ginger upon challenge inoculation. Here also, the isolates showed a profound effect on growth and yield of ginger especially by those plants bacterized with RB-11. There was no natural incidence of bacterial wilt in plants treated with RB-11 and RB-22. Upon challenge inoculation also, plants bacterized with RB-11 showed the least incidence. In general, rhizobacterial treated plants contained more amount of phenol, protein and amino acids than untreated ones. Upon challenge inoculation with the pathogen, the rate of increase of these compounds in rhizobacteria treated plants was more than that of control during different intervals of observations. A third pot culture experiment was conducted to assess the effect of rhizobacterial treatments on defense related enzymes of ginger upon challenge inoculation. Here, eight most promising ones including the reference cultures were used. In general, the study revealed more activity of peroxidase (PO), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) in rhizobacterial treated plants that too after challenge inoculation. Native PAGE analysis revealed six isoforms of PO and four isoforms of PPO in a majority of the rhizobacterial treated plants whereas only three were noticed in control. Similarly, difference in the protein profile of rhizobacterial treated plants and control was noticed. Chlorophyll, NPK and oil and oleoresin content varied among treatments where the highest was observed in rhizobacterial treated plants. An attempt was made to elucidate the molecular mechanism of induced systemic resistance (ISR) in ginger by synthesizing cDNA and was subjected to RAPD assay. However, no conclusive evidence on ISR was observed. The compatibility of eight rhizobacterial isolates including the two reference cultures with antibiotics, fungicides, insecticides and fertilizers were assessed which revealed variation in their sensitivity. Moreover, mutual compatibility of the rhizobacterial isolates and compatibility with Trichoderma spp. were also studied and it was observed that all bacterial isolates were mutually compatible. However, Pseudomonas aeruginosa, P. fluorescens (RB-66) and the reference culture of P .fluorescens (P.f2) were found incompatible with the Trichoderma spp. The promising six rhizobacteria isolates were identified based on cultural, morphological and biochemical characters and also in comparison with that of reference culture of P. fluorescens. They were tentatively identified as Pseudomonas aeruginosa (RB-22), Pseudomonas fluorescens (RB-82, RB-66, RB-11) and the remaining two, RB-144 and RB-77, as non-fluorescent Pseudomonads.