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Role of Maize Weevil, Sitophilus zeamais Motsch. on Spread of Aspergillus section flavi in Different Nepalese Maize Varieties

Role of Maize Weevil, Sitophilus zeamais Motsch. on Spread of Aspergillus section flavi in... Hindawi Advances in Agriculture Volume 2019, Article ID 7584056, 5 pages https://doi.org/10.1155/2019/7584056 Research Article Role of Maize Weevil, Sitophilus zeamais Motsch. on Spread of Aspergillus section flavi in Different Nepalese Maize Varieties K. Bhusal and D. Khanal Institute of Agriculture and Animal Sciences, Kirtipur, Kathmandu, Nepal Correspondence should be addressed to D. Khanal; dipakbabu@hotmail.com Received 10 September 2018; Revised 25 January 2019; Accepted 4 April 2019; Published 16 April 2019 Academic Editor: Innocenzo Muzzalupo Copyright © 2019 K. Bhusal and D. Khanal. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Experiments were conducted to find out the role of maize weevil, Sitophilus zeamais Motsch. on spread of green fungus, Aspergillus section afl vi, in dieff rent varieties of stored maize in laboratory in 2016. Lab experiment was conducted to find the role of weevil on spread of A. afl vus on five main varieties of maize grown at Nepal in split plot design, namely, Arun-2, Arun-4, Manakamana-1, Manakamana-3, and Rampur composite with three replications at NAST, Khumaltar, from August to September 2016. One hundred grams of each maize variety was exposed to weevil along with fungus and with fungus only to see the spread of the fungus under presence and absence of weevil. Among the tested vfi e maize varieties, the lowest infestation was observed on Rampur Composite (14.99%) while it was the highest on Manakamana-3 (87.70%). eTh highest mean infestation (75.58%) was found under weevil released condition while it was lower (62.16%) under nonreleased condition. In presence of weevil, the infestation of the fungus increased and in their absence the infestation was low which signifies the role of weevil in fungal spread. All indices indicate that Rampur composite is the best variety among the vfi e tested varieties in terms of storage under the presence of fungus and weevils. iTh s study also indicates ample scope for further study on different varieties of maize under several storage conditions. 1. Introduction moth (Sitotroga cerealella) are the most important insects in stored maize in Nepal [8, 9]. The most economically impor- Maize (Zea mays L.), belonging to the family Poaceae or tant and widely occuring postharvest losses due to insect Gramineae, is a kind of grass related to rice, wheat, barley, and pests of stored maize include the maize weevil (Sitophilus oat, ranking second in area cultivated and first in production zeamais Motsch.), a ij inch long, and reddish brown to black and productivity in order of world grain production [1]. It is snout weevil pests of stored maize include the maize weevil popularly known as queen of cereals, because of very high [10]. yield potential than any other cereals [2]. In Nepal, maize is S. zeamais Motsch. is one of the most serious, internal grown in 882,395 ha of land with an average yield of 2.43 t/ha feeding pests of maize. It is among the most destructive [3] which seems to be very low as compared to neighboring pests instored grain, especially cornintropical regions [11]. countries. Adult female of weevils causes damage by boring into the Insects are most oen ft considered as the principal cause kernel and laying eggs (ovipositing). Then, larvae and pupae of maize grain losses [4]. Postharvest loss is a measurable eat the inner parts of the kernel, resulting in a damaged quantitative, qualitative loss across the supply chain, or the kernel, and reduced grain weight [12]. The infestation elevates postharvest system, from the time of harvest till its consump- temperature and moisture content in the stored grain mass, tion [5, 6]. Postharvest loss of maize has been estimated to which can lead to fungal growth, including toxigenic species be between 15% and 26% [7]. The greatest portions of these such as Aspergillus flavus Link. [13]. losses occur in the standing crops and during storage and are Aspergillus flavus is a saprophytic soil fungus that infects mainly due to insect infestation. Among insect pests, maize and contaminates preharvest and postharvest seed crops like weevil (Sitophilus zeamais Motsch.) and Angoumois grain cereals, legumes, and oil seeds [14]. The species, A. flavus, 2 Advances in Agriculture belongs to the Genus Aspergillus, Subdivision Deuteromy- inoculation loop was dipped in the pure culture containing cotina [15]. This species grows on a wide range of agricultural A. section flavi and was released on bottles with treatment commodities that include peanuts, dried corn, millet, tree numbers 6, 7,8, 9,and 10each containing 100grams of nuts, and cotton seeds [16] and leftover foods such as rice. S. different maize varieties only. The treatments numbers 1, 2, zeamais cause extensive losses in quantity and quality of the 3, 4, and 5 dieff r with treatment 6, 7, 8, 9, and 10 in terms of grain in the field as well as in storage [17]. Numerous insect presence and absence of weevil only. The spore population species have been implicated in facilitating the dispersal of A. in inoculation loop dipped in a pure culture was calculated flavus and subsequent aflatoxin contamination [18, 19]. The by using spread plate technique [23]. Nearly an average spore spread of the green fungus in maize seed is a great problem number of 33∗10 -3 CFU was obtained. The 10 maize grains in stored maize and the metabolic activities of weevil linked in the bootless were collected aer ft 10, 12, 17, 19, 24, 26, 31, in thespread ofspores of the fungus area major reasonfor and 33 days after the inoculation of fungus and was viewed the research. under microscope to check the infestation. The infestation percentage was calculated as follows: 2. Materials and Methods Number of infected seeds 2.1. Laboratory Experiment. The experiments were con- Infestation(%)= Total number of seeds per plate (1) ducted in Natural Products Research Laboratory of Nepal Academy of Science and Technology (NAST), Khumaltar, ×100 Lalitpur, starting from June to September 2016. The resistance and susceptibility of the varieties against A. 2.1.1. Cleaning, Drying, and Sterilization of the Seed. The section flavi were categorized on the following scale given by seeds of vfi e different maize varieties, namely,Manakamana- [24]. Resistant = Less than 15% seed infection. 3, Manakamana-1, Arun-2, Arun-4, and Rampur composite Moderately resistant = 16 – 30% seed infection. collected from Nepal Agricultural Research Council (NARC), Susceptible = 31–50% seed infection. Rampur, Chitwan, were cleaned thoroughly to be free from Highly susceptible = above 50% seed infection. dust, dirt, stubbles, and foreign matter and dried in the sun In samples, undamaged and damaged grains were to reduce the moisture level of seed. The moisture level was counted and weighted separately and percentage loss was maintained from 11 to 12%. Cleaned and dried seeds were calculated as follows [25]. filled on the steel container and kept on the hot air oven at 70 C for 2 days to sterilize the seed and make it free from (UNd) –(DNu) pathogens and weevils. The pathogen was eliminated from Weight loss % = ×100 ( ) (2) ∘ ∘ maize seeds treated at 60 C for 15 days or at 70 Cfor 2days U Nd+ Nu ( ) and germination rates in most samples were not affected [20]. where U is Wt. of undamaged grains, D is Wt. of damaged 2.1.2. Mass Rearing of Weevil, Isolation, and Culture of Fungus. grain, Nd is No. of damaged grain, Nu is No. of undamaged Live insect specimens of S. zeamais were collected from Nepal grain. Agricultural Research Council (NARC), Khumaltar, Lalitpur. In this method, recommended sample size is 100-1000 Manakamana-1 variety of maize was used for insect rearing. grains. Hundred pairs of one-week-old S. zeamais were introduced The percentage of insect damaged seed was then calcu- into 2 kg grains of maize in 6.5 kg capacity Kilner jars lated [26] as follows: covered with mesh lids [21]. Infected maize cob was used for isolation of the fungus (A. section flavi ). The fungus Insect damaged percentage(%) was transferred to the Potato Dextrose Agar (PDA) media with the help of sterilized inoculation loop. The media were (3) Number of insect damaged grain = ×100 placed on the incubator at 27 C for 5 days. Isolates were Total number of grain identified as A. section flavi based on colony characteristics, strain morphology, and microscopic features according to [22]. 2.1.4. Experimental Design and Statistical Analysis. The experiment was laid out in split plot design with three repli- 2.1.3. Inoculation of the Fungus and Insect. Five pairs of wee- cations. Two factors were included in the experiment. Five vils were kept into the sterilized test tube of 5ml capacity. An varieties of maize were treated as main factor and two levels inoculation loop was dipped in the pure culture of A. section of weevil, i.e., presence and absence of weevil, were treated flavi and was introduced into the same test tube containing as subplot factors. The data of each experiment was collected the weevil and then plugged by the sterilized cotton plug. The and analyzed by suitable Microsoft sowa ft re package like R- tube was shaked so that the spores get in contact with the Stat. The mean comparisons were done by DMRT at 5% cuticle of the weevils. Those vfi e pairs of weevil treated with and 1% level of significance [27]. Regarding the sowa ft re spores of A. section flavi were released selectively on bottles programs,Microsoft word2007 was usedfor wordprocess- with treatment numbers 1, 2, 3, 4, and 5 each containing ing. MS Excel was used for data entry, tables, and graphs 100 grams of different maize varieties, while in others same preparation. Advances in Agriculture 3 Table 1: Infestation on dieff rent maize varieties by artificial inoculation of A. section afl vi in vitro recorded from August to September, 2016. A. afl vus infestation (%) Days after inoculation Maize varieties Grand Mean 10 12 17 19 24 26 31 33 days days days days days days days days 75.00 98.33 76.67 96.67 73.33 100.0 85.00 96.67 87.70 Manakamana-3 ± 7.63 ±1.67 ± 4.94 ± 3.33 ± 4.21 ± 0.00 ± 2.23 ± 2.10 (1.09) (1.50) (1.07) (1.48) (1.04) (1.55) (1.17) (1.45) 71.67 98.33 73.33 95.00 73.33 98.33 80.00 95.00 85.62 Manakamana-1 ± 6.01 ± 1.67 ± 4.94 ± 3.41 ± 3.33 ± 1.66 ± 3.64 ± 2.23 (1.02) (1.50) (1.03) (1.42) (1.03) (1.50) (1.11) (1.40) 60.00 88.33 68.33 88.33 66.67 98.33 75.00 81.67 78.33 Arun-4 ± 7.74 ± 4.77 ± 5.42 ± 4.77 ± 6.66 ± 1.66 ± 3.41 ± 4.78 (0.89) (1.28) (0.98) (1.28) (0.96) (1.50) (1.05) (1.16) 58.33 93.33 58.33 85.00 61.67 95.00 80.00 90.00 77.70 Arun-2 ± 8.72 ± 3.33 ± 7.03 ± 5.62 ± 6.01 ± 2.23 ± 3.64 ± 5.16 (0.87) (1.37) (0.87) (1.24) (0.91) (1.40) (1.11) (1.33) 3.33 23.33 3.33 21.67 5.00 18.33 20.00 25.00 14.99 Rampur composite ± 3.33 ± 4.21 ± 2.10 ± 3.07 ± 3.41 ± 5.42 ± 5.77 ± 6.70 (0.09) (0.49) (0.11) (0.47) (0.14) (0.39) (0.42) (0.48) Grand Mean 53.67 80.33 55.99 77.34 56.00 81.99 68.00 77.67 CV (%) 14.5 11.5 17.6 13.2 18.3 13.1 12.5 11.6 LSD 0.33 0.41 0.41 0.45 0.43 0.47 0.34 0.39 P- value ≤0.001 ≤0.001 ≤0.001 ≤0.001 ≤0.001 ≤0.001 ≤0.001 ≤0.001 CV: coefficient variation; LSD: least significance difference; small alphabetic superscripts are significant by Duncan’s multiple range test (p≤ 0.001); numbers in the parenthesis are the arcsine square root transformation; value after ± is the standard error of mean. Table 2: Effect of weevil on spread of Aspergillus section afl vi. on maize seeds from August to September, 2016. A. section afl vus infestation (%) Days after inoculation Weevil Grand Mean 10 days 12 days 17 days 19 days 24 days 26 days 31 days 33 days a a a a a a a a 65.33 84.67 64.67 84.00 63.33 84.67 74.00 84.00 75.58 Released ± 8.32 ± 7.85 ± 8.44 ± 7.73 ± 7.84 ± 8.21 ± 6.15 ± 6.74 (0.93) (1.33) (0.91) (1.31) (0.89) (1.32) (1.05) (1.31) b b b b b b b b 42.00 76.00 47.33 70.67 48.67 80.00 62.00 70.67 62.16 Not- released ± 6.48 ± 7.85 ± 6.43 ± 7.71 ± 6.60 ± 9.10 ± 7.18 ± 8.07 (0.65) (1.13) (0.72) (1.05) (0.74) (1.22) (0.89) (1.02) Grand Mean 53.67 80.33 56.00 77.34 56.00 82.34 68.00 77.34 CV (%) 18.4 8.9 10.4 12.1 15 7.4 9.4 12.9 LSD 0.24 0.17 0.14 0.23 0.13 0.07 0.14 0.24 P- value ≤0.001 ≤0.001 ≤0.001 ≤0.001 ≤0.01 ≤0.05 ≤0.001 ≤0.001 CV: coefficient variation; LSD: least significance difference; alphabetic superscripts are significant at p ≤0.001, p≤ 0.01 and p≤ 0.05 by Duncan’s multiple range test; numbers in the parentheses are the arcsine square root transformation; value after ± is the standard error of mean. 3. Results and Discussion ± 3.33 and 3.33± 2.10, was obtained on Rampur composite on 10 days and 17 days, respectively. Rampur composite had 3.1. Laboratory Experiments less than 15% seeds infestation with A. section flavi and all other varieties with more than 50% infestation distinguishing 3.1.1. Infestation of Dieff rent Varieties of Maize with Aspergillus between highly resistant and susceptible varieties. Similar section flavi. The infestation of different varieties of maize in findings against A. section flavi were categorized by [24]. this study is presented in Table 1. There was highly significant difference among the varieties at 10 days to 33 days. The 3.1.2. Effect of Weevil on Spread of Aspergillus section flavi on maximum infestation was obtained in Manakamana-3 at 26 Maize Storage. The effect of weevil on spread of A. flavus days, i.e., 100± 0.00, while the lowest infestation, i.e., 3.33 is presented in Table 2. There was significant difference 4 Advances in Agriculture Table 3: Damage of maize weevil, Sitophilus zeamais Motsch on maize varieties infestation with fungus under no choice condition at NAST, Lalitpur,Nepal,2016. Varieties Grain damage (Weight. basis) aer ft 30 days of treatment (%) Weevil Released Weevil Not- Released Manakamana-3 4.23± 0.65 (2.16) 0.00± 0.00 (0.70) Manakamana-1 2.94± 0.40 (1.85) 0.00± 0.00 (0.70) Arun-4 1.32± 0.34 (1.34) 0.00± 0.00 (0.70) Arun-2 1.08± 0.39 (1.24) 0.00± 0.00 (0.70) bc d Rampur composite 1.67± 0.58 (1.45) 0.00± 0.00 (0.70) Grand Mean 2.24 0.00 CV (%) 15.9 LSD 0.42 P- value ≤0.01 CV: coefficient variation; LSD: least significance difference; figures in the columns followed with alphabetic superscripts are significant at p ≤0.01 by Duncan’s multiple range test; figures in the parentheses are the square root transformation; value after ± is the standard error of mean. among the weevil and the A. flavus infestation on each the infestation of the fungus increased and in their absence condition from 10 days to 33 days of study period. The highest the infestation was low which signifies the role of weevil in infestations mean (82.34 %) was on 26 days while the lowest fungal spread. All indices indicate that Rampur composite is (53.67 %) was on 10 days. The highest mean contamination best variety among the vfi e tested varieties in terms of storage (75.58%) was found under weevil present condition while under the presence of fungus and weevils. This study also it was lower (62.16%) under absent condition. Maize weevil indicates ample scope for further study on dieff rent varieties increases the efficacy of fungus infestation in their presence of maize under several storage conditions for longer time under storage. Maize weevils cause mechanical damage and period. increased moisture content. Insect infestation usually causes increased heating and moisture content in stored grains due Data Availability to metabolic activity of the insects [28]. The number of spores The data used to support the findings of this study are avail- carried by Maize weevils, whether internally, externally, or determined from whole body extracts, increased with the able from the corresponding author upon written request. amount of time spent on the infected corn [18]. These authors reported that the maize weevil enhance growth of A. section Conflicts of Interest flavi by increasing the area susceptible to fungal growth. S. The authors declare that there are no conflicts of interest zeamais was reported to contribute significantly to increase regarding the publication of this paper. A. section flavi infection on corn ears by transporting spores and damaging corn kernels [29]. Acknowledgments 3.1.3. Damage of Maize Weevil on Maize Varieties on Weight Institute of Agriculture and Animal Science, Nepal Academy Basis. The grain damage was signicfi antly dieff rent (P ≤0.01) of Science and Technology, and United Nations Development among the tested varieties at 30 days after observations in Program are highly acknowledged for providing laboratory no-choice condition (Table 3). Aeft r 30 days of treatment facilities and financial support for this study. the highest loss was recorded on Mankamana-3 whereas the lowest loss was recorded on Arun-2, Arun-4, and Rampur References composite, respectively. Rampur composite, Arun-2, and Arun-4 were less aec ff ted by weevil. Yellow varieties were less [1] FAO, World Food and Agriculture, Food and Agriculture Orga- susceptible to weevil damage as compared to white varieties nization of United States, Rome, Italy, 2013. [30]. [2] C. Singh, Modern Techniques of Raising Field Crops,IBH Publishing Co.Pvt.Ltd, New Delhi, India, 2002. 4. Conclusions [3] MoAD, Statistical Information on Nepalese Agriculture, Agribusiness Promotion and Statistics Division, Singh Darbar, In conclusion, the maximum infestation of the fungus was Kathmandu, Nepal, 2015. obtained on Manakamana-3 variety while the lowest infesta- [4] A. R.Khosravi, M. Mansouri, A.R.Bahonar, and H.Shokri, tion was obtained on Rampur composite. The highest mean “Mycoflora of maize harvested from Iran and imported maize,” contamination was found under presence of weevil while it Pakistan Journalof BiologicalSciences, vol.10, no.24, pp. 4432– was lower under absence of weevil. Manakamana-3 variety 4437, 2007. had the highest damage while Rampur composite, Arun-2, [5] J. Aulakh and A. Regmi, “Post harvest food losses estimation,” and Arun-4 had the lowest damage. In presence of weevil, Developement of Consistent Methodology, p.6,2015. Advances in Agriculture 5 [6] T. 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Dhankuta, Nepal,” Pakhribas and Insect Pest Management, B.R Publishing Corporation, New Agriculture Center,1997. Delhi, India, 1987. [9] Y. D. Gharti Chhetri, “Efficacy of indegenous plant materials [26] P. W. Wambugu, E. O. Mathenge, Auma, and R. Havan, “Efficacy and modified storage structure to insect pests of maize during of traditional maize (Zea mays L.) seed storage methods in on farm storage,” Journal of Institute of Agriculture abd Animal western Kenya,” African Journal of Food and Agriculture,vol. 9, Science,vol.27, pp.69–76,2006. no. 4, pp. 1110–1128, 2009. [10] C. P. Rugumamu, “Assesment of post harvest technologies and [27] K. A. Gomez and A. A. Gomez, Statistical Procedures for gender relations in maize loss reduction in Pangawe village Agricultural Research, John Wiley and Sons, New York, NY, eastern Tanzania,” Tanzania Journal of Science, vol.35, pp.67– USA, 2nd edition, 1984. 76, 2012. [28] J. T. Mills, “Insect fungus association inuen fl cing seed detora- [11] J. L. Paes,L. R.D. Faroni, O. D. Dhingra, P. R.Cecon, and T. tion,” Journal of Phytopathology, pp. 330–335, 1983. A. Silva, “Insecticidal fumigation action of mustard essential [29] W. W. McMillan, N. W. Widstrom, D. M. Wilson, and R. A. oil against Sitophilus zeamais in maize grains,” Journal of Crop Hill, “Transmission by maize weevils of Aspergillus flavus and Protection,pp. 56–58, 2012. its survival on selected corn hybrids,” Journal of Economic [12] J. A. Ojo and A. A. Omoloye, “Rearing of maize weevil, Entomology,vol.36, pp. 793-794, 1980. Sitophilus zeamais on an artificial maize-cassava diet,” Journal [30] NMRP, Annual Report of National Maize Research Programme, of Insect Science, pp. 1-2, 2012. NARC, Rampur, India, 2012. [13] S. S. Chu, S. S. Du, and Z. L. Liu, “Fumigant compounds from the essential oil of Chinese Blumea balsamifera leaves against maize weevil,” Journal of Chemistry,pp. 1–7, 2013. [14] S. Amaike and N. P. Keller, Aspergillus Flavus,University of Wisconsin, Madison, Wisconsin, USA, 2011. [15] C. J. Alexopoulos and C. W. Mims, Introductory Mycology, Wiley Eastern Ltd, New Delhi, India, 1988. [16] M. Micheal and P. Ensley, Understanding Fungal (Mold) Toxins (Mycotoxins) Plant Disease, Lincoln abd the United States Department of Agriculture, Lincoln, 2007. [17] M. M. Sabbour, “Entomotoxicity assasy of two nano particle materials 1-(Al203 and TiO2) against Sitophilus oryzae under laboratory and store conditions in Egypt,” Journal of Novel Applied Sciences, vol. 1, pp. 103–108, 2012. [18] J. A. Beti, T. W. Phillips, and E. B. Smalley, “Eeff ct of maize weevils (Coleoptera: Curculionidae) on production of aflatoxin B1 by Aspergillus flavus in stored corn,” Journal of Economic Entomology, vol. 88, no. 6, pp. 1776–1782, 1995. [19] S. Mohale,J.Allotey, and B.A.Siame,“Control oftribolium confusum J. Du Val by diatomaceous earth (Protect-IT) on stored groundnut (Arachis hypogea) and Aspergillus flavus Link spore dispersal,” African Journal of Food Agriculture Nutrition and Developement,vol. 10,no.6, pp. 2679–2694,2010. [20] R. M. Clear, S. K. Patrick, R. Wallis, and T. K. Turkington, “Eeff ct of dry heat treatment on seed-borne Fusarium graminearum and other cereal pathogens,” Canadian Journal of Plant Pathol- ogy,vol.24,no.4,pp.489–498, 2002. [21] J. A. Ojo and A. A. Amoloye, “Development and life history of sitophilus zeamais (coleoptera: curculionidae) on cereal crops,” Advances in Agriculture,pp. 1–8, 2016. [22] M. A. Klich and L. S. Lee, “Seed viability and aflatoxin pro- duction in individual cotton seed natirally contaminated with Aspergillus flavus,” Journal of the American Oil Chemists’ Society,p. 545, 2002. [23] B.Hunsinger, B.A.Kamp,C.Kumala,and R.Bohm, ¨ “Com- parison of the spread plate technique and the MPN-technique The Scientific International Journal of Journal of Veterinary Medicine Food Science Botany Scientica International World Journal Hindawi Hindawi Hindawi Hindawi Hindawi Publishing Corporation Hindawi www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 http://www www.hindawi.com .hindawi.com V Volume 2018 olume 2013 International Journal of International Journal of Microbiology Cell Biology Hindawi Hindawi www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 International Journal of International Journal of Agronomy Ecology Submit your manuscripts at www.hindawi.com Hindawi Hindawi www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 Journal of International Journal of Biotechnology International Journal of Nutrition and Plant Genomics Research International Forestry Research Psyche Metabolism Hindawi Hindawi Hindawi Hindawi Hindawi www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 Applied & Environmental International Journal of Advances in International Journal of BioMed Soil Science Genomics Agriculture Biodiversity Research International Hindawi Hindawi Hindawi Volume 2018 Hindawi Hindawi www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Advances in Agriculture Hindawi Publishing Corporation

Role of Maize Weevil, Sitophilus zeamais Motsch. on Spread of Aspergillus section flavi in Different Nepalese Maize Varieties

Advances in Agriculture , Volume 2019 – Apr 16, 2019

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Abstract

Hindawi Advances in Agriculture Volume 2019, Article ID 7584056, 5 pages https://doi.org/10.1155/2019/7584056 Research Article Role of Maize Weevil, Sitophilus zeamais Motsch. on Spread of Aspergillus section flavi in Different Nepalese Maize Varieties K. Bhusal and D. Khanal Institute of Agriculture and Animal Sciences, Kirtipur, Kathmandu, Nepal Correspondence should be addressed to D. Khanal; dipakbabu@hotmail.com Received 10 September 2018; Revised 25 January 2019; Accepted 4 April 2019; Published 16 April 2019 Academic Editor: Innocenzo Muzzalupo Copyright © 2019 K. Bhusal and D. Khanal. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Experiments were conducted to find out the role of maize weevil, Sitophilus zeamais Motsch. on spread of green fungus, Aspergillus section afl vi, in dieff rent varieties of stored maize in laboratory in 2016. Lab experiment was conducted to find the role of weevil on spread of A. afl vus on five main varieties of maize grown at Nepal in split plot design, namely, Arun-2, Arun-4, Manakamana-1, Manakamana-3, and Rampur composite with three replications at NAST, Khumaltar, from August to September 2016. One hundred grams of each maize variety was exposed to weevil along with fungus and with fungus only to see the spread of the fungus under presence and absence of weevil. Among the tested vfi e maize varieties, the lowest infestation was observed on Rampur Composite (14.99%) while it was the highest on Manakamana-3 (87.70%). eTh highest mean infestation (75.58%) was found under weevil released condition while it was lower (62.16%) under nonreleased condition. In presence of weevil, the infestation of the fungus increased and in their absence the infestation was low which signifies the role of weevil in fungal spread. All indices indicate that Rampur composite is the best variety among the vfi e tested varieties in terms of storage under the presence of fungus and weevils. iTh s study also indicates ample scope for further study on different varieties of maize under several storage conditions. 1. Introduction moth (Sitotroga cerealella) are the most important insects in stored maize in Nepal [8, 9]. The most economically impor- Maize (Zea mays L.), belonging to the family Poaceae or tant and widely occuring postharvest losses due to insect Gramineae, is a kind of grass related to rice, wheat, barley, and pests of stored maize include the maize weevil (Sitophilus oat, ranking second in area cultivated and first in production zeamais Motsch.), a ij inch long, and reddish brown to black and productivity in order of world grain production [1]. It is snout weevil pests of stored maize include the maize weevil popularly known as queen of cereals, because of very high [10]. yield potential than any other cereals [2]. In Nepal, maize is S. zeamais Motsch. is one of the most serious, internal grown in 882,395 ha of land with an average yield of 2.43 t/ha feeding pests of maize. It is among the most destructive [3] which seems to be very low as compared to neighboring pests instored grain, especially cornintropical regions [11]. countries. Adult female of weevils causes damage by boring into the Insects are most oen ft considered as the principal cause kernel and laying eggs (ovipositing). Then, larvae and pupae of maize grain losses [4]. Postharvest loss is a measurable eat the inner parts of the kernel, resulting in a damaged quantitative, qualitative loss across the supply chain, or the kernel, and reduced grain weight [12]. The infestation elevates postharvest system, from the time of harvest till its consump- temperature and moisture content in the stored grain mass, tion [5, 6]. Postharvest loss of maize has been estimated to which can lead to fungal growth, including toxigenic species be between 15% and 26% [7]. The greatest portions of these such as Aspergillus flavus Link. [13]. losses occur in the standing crops and during storage and are Aspergillus flavus is a saprophytic soil fungus that infects mainly due to insect infestation. Among insect pests, maize and contaminates preharvest and postharvest seed crops like weevil (Sitophilus zeamais Motsch.) and Angoumois grain cereals, legumes, and oil seeds [14]. The species, A. flavus, 2 Advances in Agriculture belongs to the Genus Aspergillus, Subdivision Deuteromy- inoculation loop was dipped in the pure culture containing cotina [15]. This species grows on a wide range of agricultural A. section flavi and was released on bottles with treatment commodities that include peanuts, dried corn, millet, tree numbers 6, 7,8, 9,and 10each containing 100grams of nuts, and cotton seeds [16] and leftover foods such as rice. S. different maize varieties only. The treatments numbers 1, 2, zeamais cause extensive losses in quantity and quality of the 3, 4, and 5 dieff r with treatment 6, 7, 8, 9, and 10 in terms of grain in the field as well as in storage [17]. Numerous insect presence and absence of weevil only. The spore population species have been implicated in facilitating the dispersal of A. in inoculation loop dipped in a pure culture was calculated flavus and subsequent aflatoxin contamination [18, 19]. The by using spread plate technique [23]. Nearly an average spore spread of the green fungus in maize seed is a great problem number of 33∗10 -3 CFU was obtained. The 10 maize grains in stored maize and the metabolic activities of weevil linked in the bootless were collected aer ft 10, 12, 17, 19, 24, 26, 31, in thespread ofspores of the fungus area major reasonfor and 33 days after the inoculation of fungus and was viewed the research. under microscope to check the infestation. The infestation percentage was calculated as follows: 2. Materials and Methods Number of infected seeds 2.1. Laboratory Experiment. The experiments were con- Infestation(%)= Total number of seeds per plate (1) ducted in Natural Products Research Laboratory of Nepal Academy of Science and Technology (NAST), Khumaltar, ×100 Lalitpur, starting from June to September 2016. The resistance and susceptibility of the varieties against A. 2.1.1. Cleaning, Drying, and Sterilization of the Seed. The section flavi were categorized on the following scale given by seeds of vfi e different maize varieties, namely,Manakamana- [24]. Resistant = Less than 15% seed infection. 3, Manakamana-1, Arun-2, Arun-4, and Rampur composite Moderately resistant = 16 – 30% seed infection. collected from Nepal Agricultural Research Council (NARC), Susceptible = 31–50% seed infection. Rampur, Chitwan, were cleaned thoroughly to be free from Highly susceptible = above 50% seed infection. dust, dirt, stubbles, and foreign matter and dried in the sun In samples, undamaged and damaged grains were to reduce the moisture level of seed. The moisture level was counted and weighted separately and percentage loss was maintained from 11 to 12%. Cleaned and dried seeds were calculated as follows [25]. filled on the steel container and kept on the hot air oven at 70 C for 2 days to sterilize the seed and make it free from (UNd) –(DNu) pathogens and weevils. The pathogen was eliminated from Weight loss % = ×100 ( ) (2) ∘ ∘ maize seeds treated at 60 C for 15 days or at 70 Cfor 2days U Nd+ Nu ( ) and germination rates in most samples were not affected [20]. where U is Wt. of undamaged grains, D is Wt. of damaged 2.1.2. Mass Rearing of Weevil, Isolation, and Culture of Fungus. grain, Nd is No. of damaged grain, Nu is No. of undamaged Live insect specimens of S. zeamais were collected from Nepal grain. Agricultural Research Council (NARC), Khumaltar, Lalitpur. In this method, recommended sample size is 100-1000 Manakamana-1 variety of maize was used for insect rearing. grains. Hundred pairs of one-week-old S. zeamais were introduced The percentage of insect damaged seed was then calcu- into 2 kg grains of maize in 6.5 kg capacity Kilner jars lated [26] as follows: covered with mesh lids [21]. Infected maize cob was used for isolation of the fungus (A. section flavi ). The fungus Insect damaged percentage(%) was transferred to the Potato Dextrose Agar (PDA) media with the help of sterilized inoculation loop. The media were (3) Number of insect damaged grain = ×100 placed on the incubator at 27 C for 5 days. Isolates were Total number of grain identified as A. section flavi based on colony characteristics, strain morphology, and microscopic features according to [22]. 2.1.4. Experimental Design and Statistical Analysis. The experiment was laid out in split plot design with three repli- 2.1.3. Inoculation of the Fungus and Insect. Five pairs of wee- cations. Two factors were included in the experiment. Five vils were kept into the sterilized test tube of 5ml capacity. An varieties of maize were treated as main factor and two levels inoculation loop was dipped in the pure culture of A. section of weevil, i.e., presence and absence of weevil, were treated flavi and was introduced into the same test tube containing as subplot factors. The data of each experiment was collected the weevil and then plugged by the sterilized cotton plug. The and analyzed by suitable Microsoft sowa ft re package like R- tube was shaked so that the spores get in contact with the Stat. The mean comparisons were done by DMRT at 5% cuticle of the weevils. Those vfi e pairs of weevil treated with and 1% level of significance [27]. Regarding the sowa ft re spores of A. section flavi were released selectively on bottles programs,Microsoft word2007 was usedfor wordprocess- with treatment numbers 1, 2, 3, 4, and 5 each containing ing. MS Excel was used for data entry, tables, and graphs 100 grams of different maize varieties, while in others same preparation. Advances in Agriculture 3 Table 1: Infestation on dieff rent maize varieties by artificial inoculation of A. section afl vi in vitro recorded from August to September, 2016. A. afl vus infestation (%) Days after inoculation Maize varieties Grand Mean 10 12 17 19 24 26 31 33 days days days days days days days days 75.00 98.33 76.67 96.67 73.33 100.0 85.00 96.67 87.70 Manakamana-3 ± 7.63 ±1.67 ± 4.94 ± 3.33 ± 4.21 ± 0.00 ± 2.23 ± 2.10 (1.09) (1.50) (1.07) (1.48) (1.04) (1.55) (1.17) (1.45) 71.67 98.33 73.33 95.00 73.33 98.33 80.00 95.00 85.62 Manakamana-1 ± 6.01 ± 1.67 ± 4.94 ± 3.41 ± 3.33 ± 1.66 ± 3.64 ± 2.23 (1.02) (1.50) (1.03) (1.42) (1.03) (1.50) (1.11) (1.40) 60.00 88.33 68.33 88.33 66.67 98.33 75.00 81.67 78.33 Arun-4 ± 7.74 ± 4.77 ± 5.42 ± 4.77 ± 6.66 ± 1.66 ± 3.41 ± 4.78 (0.89) (1.28) (0.98) (1.28) (0.96) (1.50) (1.05) (1.16) 58.33 93.33 58.33 85.00 61.67 95.00 80.00 90.00 77.70 Arun-2 ± 8.72 ± 3.33 ± 7.03 ± 5.62 ± 6.01 ± 2.23 ± 3.64 ± 5.16 (0.87) (1.37) (0.87) (1.24) (0.91) (1.40) (1.11) (1.33) 3.33 23.33 3.33 21.67 5.00 18.33 20.00 25.00 14.99 Rampur composite ± 3.33 ± 4.21 ± 2.10 ± 3.07 ± 3.41 ± 5.42 ± 5.77 ± 6.70 (0.09) (0.49) (0.11) (0.47) (0.14) (0.39) (0.42) (0.48) Grand Mean 53.67 80.33 55.99 77.34 56.00 81.99 68.00 77.67 CV (%) 14.5 11.5 17.6 13.2 18.3 13.1 12.5 11.6 LSD 0.33 0.41 0.41 0.45 0.43 0.47 0.34 0.39 P- value ≤0.001 ≤0.001 ≤0.001 ≤0.001 ≤0.001 ≤0.001 ≤0.001 ≤0.001 CV: coefficient variation; LSD: least significance difference; small alphabetic superscripts are significant by Duncan’s multiple range test (p≤ 0.001); numbers in the parenthesis are the arcsine square root transformation; value after ± is the standard error of mean. Table 2: Effect of weevil on spread of Aspergillus section afl vi. on maize seeds from August to September, 2016. A. section afl vus infestation (%) Days after inoculation Weevil Grand Mean 10 days 12 days 17 days 19 days 24 days 26 days 31 days 33 days a a a a a a a a 65.33 84.67 64.67 84.00 63.33 84.67 74.00 84.00 75.58 Released ± 8.32 ± 7.85 ± 8.44 ± 7.73 ± 7.84 ± 8.21 ± 6.15 ± 6.74 (0.93) (1.33) (0.91) (1.31) (0.89) (1.32) (1.05) (1.31) b b b b b b b b 42.00 76.00 47.33 70.67 48.67 80.00 62.00 70.67 62.16 Not- released ± 6.48 ± 7.85 ± 6.43 ± 7.71 ± 6.60 ± 9.10 ± 7.18 ± 8.07 (0.65) (1.13) (0.72) (1.05) (0.74) (1.22) (0.89) (1.02) Grand Mean 53.67 80.33 56.00 77.34 56.00 82.34 68.00 77.34 CV (%) 18.4 8.9 10.4 12.1 15 7.4 9.4 12.9 LSD 0.24 0.17 0.14 0.23 0.13 0.07 0.14 0.24 P- value ≤0.001 ≤0.001 ≤0.001 ≤0.001 ≤0.01 ≤0.05 ≤0.001 ≤0.001 CV: coefficient variation; LSD: least significance difference; alphabetic superscripts are significant at p ≤0.001, p≤ 0.01 and p≤ 0.05 by Duncan’s multiple range test; numbers in the parentheses are the arcsine square root transformation; value after ± is the standard error of mean. 3. Results and Discussion ± 3.33 and 3.33± 2.10, was obtained on Rampur composite on 10 days and 17 days, respectively. Rampur composite had 3.1. Laboratory Experiments less than 15% seeds infestation with A. section flavi and all other varieties with more than 50% infestation distinguishing 3.1.1. Infestation of Dieff rent Varieties of Maize with Aspergillus between highly resistant and susceptible varieties. Similar section flavi. The infestation of different varieties of maize in findings against A. section flavi were categorized by [24]. this study is presented in Table 1. There was highly significant difference among the varieties at 10 days to 33 days. The 3.1.2. Effect of Weevil on Spread of Aspergillus section flavi on maximum infestation was obtained in Manakamana-3 at 26 Maize Storage. The effect of weevil on spread of A. flavus days, i.e., 100± 0.00, while the lowest infestation, i.e., 3.33 is presented in Table 2. There was significant difference 4 Advances in Agriculture Table 3: Damage of maize weevil, Sitophilus zeamais Motsch on maize varieties infestation with fungus under no choice condition at NAST, Lalitpur,Nepal,2016. Varieties Grain damage (Weight. basis) aer ft 30 days of treatment (%) Weevil Released Weevil Not- Released Manakamana-3 4.23± 0.65 (2.16) 0.00± 0.00 (0.70) Manakamana-1 2.94± 0.40 (1.85) 0.00± 0.00 (0.70) Arun-4 1.32± 0.34 (1.34) 0.00± 0.00 (0.70) Arun-2 1.08± 0.39 (1.24) 0.00± 0.00 (0.70) bc d Rampur composite 1.67± 0.58 (1.45) 0.00± 0.00 (0.70) Grand Mean 2.24 0.00 CV (%) 15.9 LSD 0.42 P- value ≤0.01 CV: coefficient variation; LSD: least significance difference; figures in the columns followed with alphabetic superscripts are significant at p ≤0.01 by Duncan’s multiple range test; figures in the parentheses are the square root transformation; value after ± is the standard error of mean. among the weevil and the A. flavus infestation on each the infestation of the fungus increased and in their absence condition from 10 days to 33 days of study period. The highest the infestation was low which signifies the role of weevil in infestations mean (82.34 %) was on 26 days while the lowest fungal spread. All indices indicate that Rampur composite is (53.67 %) was on 10 days. The highest mean contamination best variety among the vfi e tested varieties in terms of storage (75.58%) was found under weevil present condition while under the presence of fungus and weevils. This study also it was lower (62.16%) under absent condition. Maize weevil indicates ample scope for further study on dieff rent varieties increases the efficacy of fungus infestation in their presence of maize under several storage conditions for longer time under storage. Maize weevils cause mechanical damage and period. increased moisture content. Insect infestation usually causes increased heating and moisture content in stored grains due Data Availability to metabolic activity of the insects [28]. The number of spores The data used to support the findings of this study are avail- carried by Maize weevils, whether internally, externally, or determined from whole body extracts, increased with the able from the corresponding author upon written request. amount of time spent on the infected corn [18]. These authors reported that the maize weevil enhance growth of A. section Conflicts of Interest flavi by increasing the area susceptible to fungal growth. S. The authors declare that there are no conflicts of interest zeamais was reported to contribute significantly to increase regarding the publication of this paper. A. section flavi infection on corn ears by transporting spores and damaging corn kernels [29]. Acknowledgments 3.1.3. Damage of Maize Weevil on Maize Varieties on Weight Institute of Agriculture and Animal Science, Nepal Academy Basis. The grain damage was signicfi antly dieff rent (P ≤0.01) of Science and Technology, and United Nations Development among the tested varieties at 30 days after observations in Program are highly acknowledged for providing laboratory no-choice condition (Table 3). Aeft r 30 days of treatment facilities and financial support for this study. the highest loss was recorded on Mankamana-3 whereas the lowest loss was recorded on Arun-2, Arun-4, and Rampur References composite, respectively. Rampur composite, Arun-2, and Arun-4 were less aec ff ted by weevil. Yellow varieties were less [1] FAO, World Food and Agriculture, Food and Agriculture Orga- susceptible to weevil damage as compared to white varieties nization of United States, Rome, Italy, 2013. [30]. [2] C. Singh, Modern Techniques of Raising Field Crops,IBH Publishing Co.Pvt.Ltd, New Delhi, India, 2002. 4. 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