To examine the storage losses, 25 samples of chick pea seeds were collected from grocery stores of Gurgaon and Gorakhpur for examination of associated fungi(Fig.1) and insects. Seventeen fungal species viz., Alternaria alternata, Aspergillus flavus, A. niger, A. ochraceous,A.oryzae, A. terreus, A. sydowi, Chaetomium globosum, Colletotrichum dematium, Curvularialunata, Fusarium moniliforme, F. oxysporum, F. solani, Penicillium italicum, Rhizopus stolonifer, Rhizoctonia solani, Sclerotium rolfsii were found to be associated in stored chick pea (Cicer arietinum L.) seeds on the basis of microscopic and macroscopic observations.The agar plate method of study revealed presence of 17 fungi while blotter had 13 fungal species. In these Aspergillus flavus, A. niger, Fusarium oxysporum had dominance in terms of per cent occurrence showing 55.3,50.4,40.1.0% on blotter and 51.0,52.0,50.0 % in agar plates, respectively(Table 1).
The insect analysis revealed presence of only one species of
Bruchid (Callosobruchus chinensis L) in all the 25 samples(Fig.2).The adult beetle is chockalate coloured ,oval shaped,3 - 4 mm long.The body is pointed towards front.More attacks were observed during July-August in the stores.This showed feeding by making a small hole.There was presence of white eggs in Infested seeds on the surface and round exit holes with the 'flap' of seed coat.
Time to time fungal species were observed by various workers viz.,Nine fungal species -Alternaria alternata, Aspergillus flavus, Aspergillus niger, Curvularialunata [Cochliobolus lunatus], Fusarium moniliforme [Gibberella moniliformis], Helminthosporium sativum [Cochliobolus sativum], Mucor sp., Penicillium notatum and Rhizopus nigricans [R. stolonifer] fromKumarganj, Faizabad India 26(Singh et al.,2005); thirty fungal species were isolated from these seeds and most abundant-among these, were Alternaria alternata, Chaetomium spp., Penicillium citrinum, Aspergillus niger, A. flavus, Rhizopus nigricans, Fusarium oxysporum 27(Zaidi and Pathak,2013); many fungal species viz. Alternaria alternata, Aspergillus terrus, A. flavus, A. fumigatus, A. niger, Botrytis sp., Cladosporium, Curvularialunata, Fusarium solani, F. moniliforme, F. oxysporum, Macrophominaphaseolina, Penicillium notatum, Rhizoctonia sp. and Rhizopus nigricans 28(Ghangoaker and Kshirsagar ,2013);seven fungal species such as Alternaria alternata, Aspergillus flavus , A. niger, A. fumigatus, Curvularialunata, Fusarium monoliforme and Rhizoctonia solani29 (Singh, 2014); Thirteen different fungi like Actinomucor repens, Alternaria alternata, Aspergillus flavus, A. fumigatus, A. niger, A. ochraceus, Cladosporium sp., Fusarium oxysporum, Fusarium sp., Mucor varians, Penicillium notatum, Phomaherbarum, Rhizopus stolonifer 30( Sontakke and Hedawoo ,2014); Aspergillus niger (83%), Fusarium solani (79%), Aspergillus flavus (75%), Alternaria alternata (60%) and Aspergillus nidulans (57%). Minimum seed borne fungi were reported to beRhizopus stolonifer, and Fusarium oxysporum 31(Padmaja et al.,2015);. 14 fungi comprising 12 genera namely Aspergillus flavus, Aspergillus niger, Aspergillus sp, Botryodiplodiatheobromae, Botryti cinerea, Curvularialunata, Chaetomium globosum, Cladosporium sp., Colletotrichum dematium, Fusarium oxysporum, Macrophominaphasaelina, Rhizoctonia solani, Rhizopus stolonifer and Stemphylium sarciniforme32(Hossain et al.,2018); Nine fungal species namely Alternaria alternata (Fr.) Keissl., Aspergillus flavus Link, Aspergillus niger van Tieghem, Aspergillus oryzae (Ahlburg) E. Cohn, Fusarium oxysporumSchlecht. Emend. Snyder & Hansen, Macrophominaphaseolina (Tassi) Goid., Mucor sp., Penicillium italicumWehmer and Sclerotium rolfsii Sacc 33 (Javid et al.,2019),A. flavus, A.fumigatus and A.niger, Drechslera tetramera, Fusarium moniliforme, Rhizopus stolonifer34.The fungal species viz.,Chaetomium globosum, Colletotrichum dematium,Penicilliumitalicum, Sclerotium rolfsii(could)did not grow on blotters.A study 26(Singh et al.,2005) revealed that P. notatum developed on agar plate only 26(Singh et al.,2005).This may be because of fungal nutritional requirements.The no of fungal species were reduced in surface sterilized seeds which indicates that many of the fungi were located on seed coat.The sterile chick pea seeds with 0.1% Mercuric chloride projected less per cent occurrence of fungal colonies(Table 1).
The seed mycoflora devalue the seed quality, reduce its nutritional value and cause a germination failure of the seedlings and of the crop raised from such infected seeds 27( Zaidi and Pathak,2013). Based on the data in the Table 2,A. flavus, A. niger ,F.oxysporum and the insect species – C. chinensis played an important role in weight loss,seed germination,carbohydrate and protein content. A.flavus inoculated seeds showed 50%, A. niger 53%,F.oxysporum 40% while insect inoculated 39% while control set without fungus or insect inoculation showed 90-98% seed germination.The seeds inoculated with fungi/insects showed 30.1,29.4,28.7,27.4 % carbohydrate content while control set showed 61%.It is evident from Table 2that fungal/insect inoculated chickpea seeds after 21 days of storage had 10.3,9.3.10.4,8.5%protein content while control seeds were having 20% protein content. On account of wide occurrence and their pathogenicity these were selected as test organisms.
Storage conditions and containers are significant in preservation and maintenance of healthy seed during post harvest period 35 (Kandhare,2018).The variation in fungal species may be due to different isolation periods and different storage containers. The isolated species in present investigation are different may be due to different climatic conditions.
The yield of essential oil by hydrodistillation of Lavang(clove bud) was 13.57 % while the leaves of A.graveolens L. was 1.05% respectively. Fumigant fungitoxic action study of Both oils at 500 ppm(0.025ml) concentration showed 100%inhibition against Aspergillus flavus, A. niger, Fusarium oxysporum separately.
It has been extensively reported that plant based essential oils are alternative to synthetic pesticides since they are known to have minimal environmental impact and danger to consumers 36-38 (Alpaet al., 2010; Katochet al.,2012, Sharma et al.,2016).
For prescription an appropriate dose for the determination of MIC(minimum inhibitory concentration)is necessary. In the study,Lavang oil displayed highest antifungal activity against Aspergillus flavus, A. niger, Fusarium oxysporum and completetely inhibited mycelial growth(100%)of fungal speciesat 300ppm and found fungicidal at 400ppm.Sowa oil showed complete inhibition at 400ppm and found fungicidal at 500ppm.While mixture oil showed complete inhibition (100%) and fungicidal action at 400ppm against domiant fungi- Aspergillus flavus, A. niger, Fusarium oxysporum(Table 3). Both Lavang and sowa oil showed 100 per cent repellent activity at 0.01 ml dose.This was also cidal at 0.02ml dose against test insect-Callosobruchus chinensis.Mixture of both oils was cidal at 0.02ml dose.
The study on mixture of oils for antifungal spectrum revealed it knocked all seventeen fungi(A. alternata, Aspergillus flavus, A. niger, A. ochraceous,A.oryzae, A. terreus, A. sydowi, Chaetomium globosum, Colletotrichum dematium, Curvularia lunata, Fusarium moniliforme, F. oxysporum, F. solani, Penicillium italicum, Rhizopus stolonifer, Rhizoctonia solani, Sclerotium rolfsii )at 500ppm concentration.It showed 70-93% inhibitory activity against all 17 fungi at 300ppm isolated from chickpea seeds(Table 4).The oil mixture’s activity was not affected by autoclaving at 15 lb/psi at 120° C.It (safely) inhibited fungal growth even after storage of oil upto150 days(Table 5).
In this study,Lavang oil showed MIC 300ppm,Sowa oil 400ppm while the mixture had 400ppm against Aspergillus flavus, A. niger, Fusarium oxysporum.Previous studies depicted that there is a marked variation in the MIC of different plant oils at different concentrations againstA.niger viz.,Ocimumadscendensat 200ppm 39(Asthana and Singh,1981); Syzygiumaromaticum(L.)Merrilland Perry 200ppm 40(Khan,1993); Cedrus deodara at 1000ppm and Trachyspermum ammi at 500ppm 41(Singh and Tripathi,1999);Adhatoda vasicaat 500ppm 42(Kumar,2014).Cuminumcyminumat 400ppm 25(Kumar,2016);Tinospora cordifolia at 400ppm 43(Kumar et al.,2020).The differences in the MIC of various plant oils may be due totheir being different in chemical constituents or other chemical or physical parameters.
A fungicide must retain its fungitoxicity even after autoclaving44(Wellman,1967).The fungitoxicity of oil mixture (Lavang and sowa leaf oil )retained the fungitoxicity(Table 4) even after autoclaving(15lbs/psi)like that of Ageratum conyzoides 45(Dixit et al.,1995); Nardostachysjatamansi 46(Mishra et al.,1995);Adhatodavasica oil 42 (Kumar,2014); Cuminum cyminum 25(Kumar,2016)and Tinospora cordifolia 47(Kumar et al.,2020).This quality of oil will facilitate the isolation of their constituents in active state.It is also evident that a fungicide must be able to retain its activity for a long storage period.The fungitoxic factor in the oil of Adenocalyma allicea was lost within 21 day of storage 48(Chaturvedi,1979) while retained for longer periods in the oils of Ageratum conyzoides 45(Dixit et al.,1995) ; Trachyspermum ammi 41(Singh and Tripathi,1999);Adhatoda vasica 42(Kumar,2014);Cuminum cyminum 25(Kumar,2016)and Tinospora cordifolia 47(Kumar,2017;). The antifungal Toxicity of mixture oil (Lavang and sowa leaf oil )was not affected by storage upto 150days.It may be suggested that the oil mixture (Lavang and sowa leaf oil )can be safely stored for long periods(150 days) without having any adverse effecton toxicity.
The physiochemical properties of the Lavang and sowa leaf oil are recorded in Table6. GC-MS analysis of Lavang oil revealed major components viz., 75.63 %eugenol, 12.67 %caryophyllene, 8.34% eugenyl acetate and 1.63% α-humulene while the minor components were 0.03% β-elemene,0.06% α-cadinene and 0.07% ledol. The GC-MS analysis of sowa leaf oil showed presence of major constituents viz.,25.14% apiole ,12.79% o-cymene ,15.67% α-thujene, 7.13 % β-phellandrene while minor components viz., 4.87% exo-2-hydroxycineol ,4.13% limonene ,2.83% 3- isoproyl-4-methyl-1-pentyn-3-ol ,2.14% myristicine (2.16%) and 2.13 % dithydroumbell the oil ulone .
The study also showed that mixture oil (Lavang and sowa leaf oil )was more effective than salphos. It is evident from Table 7,that salphos controlled a maximum of 3 fungi while the oil mixture showed complete seed protection showing growth of no fungi and insect-Callosobruchus chinensis even after 150 days of storage.It shows its potential efficacy as the Seed Protectant as it is able to protect all 17 fungi.(Table 7). Hence it appears that mixture oil (Lavang and sowa leaf oil )increases the chickpea seeds shelf life. The seeds which served as control, showed proliferation of all 17 fungal species after 150 days of storage.Our study revealed that mixture oil (Lavang and sowa leaf oil )was more fungitoxic than salphos used in our experiments.No insect species was observed in the oil mixture treated seeds in both containers while it was present in the sulphos treated and control sets. The antifungal and insect repellent propertyof this mixture oil (Lavang and sowa leaf oil )suggests to exploit it as an ideal protectant of chickpea seeds during storage.
After 150 days of storage,germination tests revealed 90-95% seed germination in oils mixture’s treated sets,salphos treated sets revealed 60-65% while control set revealed 50-55% seed germination from seeds of both containers. The mixture oil showed no adverse effect on seedling growth and general health and morphology of plants.Thus,themixture oil (Lavang and sowa leaf oil )showed great potential as protective agent for chickpea seeds against spoilage by fungi and insects during storage.