Đokić et al. 2023, Biologica Nyssana 14(1) 14 (1) June 2023: 57-63 DOI: 10.5281/zenodo.8027182 Impact of Cuscuta spp. seed size variability on machine operation during seed finishing of natural alfalfa seeds Original Article Dragoslav Đokić Faculty of Agriculture, University of Niš, Kosančićeva 4, Kruševac, Serbi djdragosla01@gmail.com (corresponding author) Violeta Oro Institute for Plant Protection and Environment, 11000 Belgrade, Teodora Drajzera 9, Serbia Ratibor Štrbanović Institute for Plant Protection and Environment, 11000 Belgrade, Teodora Drajzera 9, Serbia Dobrivoj Poštić Institute for Plant Protection and Environment, 11000 Belgrade, Teodora Drajzera 9, Serbia Jasmina Milenković Institute for Forage Crops, 37251 Globoder-Kruševac, Serbia Jasmina Knežević Faculty of Agriculture, University of Priština - Kosovska Mitrovica, Lešak 38219, Kopaonička bb, Serbia Rade Stanisavljević Institute for Plant Protection and Environment, 11000 Belgrade, Teodora Drajzera 9, Serbia stanisavljevirade@gmail.com (corresponding author) Received: December 05, 2022 Revised: March 02, 2023 Accepted: March 07, 2023 Abstract: The alfalfa seeds intended for marketing must not contain any seeds of the dodder (Cuscuta spp.), because it is a quarantine weed in Serbia and the world. Cuscuta spp. are frequently found in natural alfalfa seeds; nevertheless and because of this, the seed must be processed, i.e. cleaned of weeds. The paper presents a study of seed size variability of Cuscuta spp. of four seed lots of natural alfalfa seeds originating from Banat, Serbia. From each of the four examined seed lots of natural alfalfa seeds, a difference in seed size was determined for 30 seeds of Cuscuta spp. per lot. The difference in seed length of Cuscuta spp. was 371 µm, and the difference in seed width was 330 µm. The coefficient of variation varied from CV=8.356% to 12.00% for seed width; and from CV=9.383% to 10.69% for seed length. Seed size of Cuscuta spp. did not significantly affect the efficiency of magnetic separator work in extracting Cuscuta spp. seeds from natural alfalfa seeds. Key words: Cuscuta spp., variability, seed width and seed length, seed processing Apstrakt: Uticaj varijabilnosti veličine semena Cuscuta spp. na rad mašina pri doradi naturalnog semena lucerke Seme viline kosice (Cuscuta spp.) je karantinski korov u Srbiji i svetu i zato se u semenu lucerke koje je namenjeno prodaji, ne sme naći seme korova Cuscuta spp. Međutim, seme Cuscuta spp. se relativno često može naći u naturalnom semenu lucerke i zbog toga se seme mora doraditi, odnosno očistiti od korova. U radu je prikazano ispitivanje varijabilnosti veličine semena Cuscuta spp. koja je izdvojena iz naturalnog semena četiri partije semena lucerke poreklom iz Banata, Srbija. Na po 30 semena Cuscuta spp., koja su izdvojena iz svake od četiri ispitivane partije naturalnog semena lucerke, uvrđena je varijabilnost za veličini semena. Razlika po dužini semena Cuscuta spp. je bila 371 µm, a po širini 330 µm. Izraženo kroz varijabilnost, koeficijent varijacije se kretao od CV=8.356 do 12.0% za širinu semena i od CV=9.383 do 10.69% za dužinu semena. Veličina semena Cuscuta spp. nije uticala značajno na efikasnost rada magnetnog separatora prilikom izdvajanja iz naturalnog semena lucerke Ključne reči: Cuscuta spp., varijabilnost, širina i dužina semena, proces dorade Introduction Dodder (Cuscuta spp.) belongs to the genus Convolvulaceae which includes about 200 mostly parasitic species (Costea et al., 2015 a,b). It is a parasitic plant that causes significant economic damage around the world (Dawson et al., 1994; Costea & Tardif, 2006; Sarić-Krsmanović et al., 2020). It is especially harmful in alfalfa seed crops (Dawson et al., 1994; Sarić-Krsmanović et al., 2020). Dodder seed causes damage by extracting nutrients from the parent plant; thus, the parent plant weakens, becomes exhausted and lags behind in development. About ten species of Cuscuta spp. were detected in Serbia in the 1960s and 1970s, and they can cause significant damage to agricultural crops (Stojanović et al., 1973), with a particularly large problem in alfalfa (Mijatović & Stojanović, 1968). In agronomic practice, it is considered that it is best to control Cuscuta spp. in the seed crop before harvesting. Nevertheless, it happens that alfalfa seed crops often contain Cuscuta spp. According to Janjic et al. (2005), in Serbia, protection from Cuscuta spp. began with weed control in crops that had © 2023 Đokić et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 57 already been infected. However, a better approach is to apply preventive measures that should be the basic way of weed control. The properties of the dodder seed, and above all, its vitality in the soil, enables the weeded parcels to become a source of further infection during the next ten or more years. Therefore, the basic strategy in the fight must be to prevent infestation of parcels (Janjić et al., 2005). Regardless of the numerous ways of testing and experience in biology and agronomic practice to study its characteristics and control to this day, it is widespread and causes significant damage in the world (Abdel-Khalik, 2006; Córdoba et al., 2021) and Serbia (Sarić-Krsmanović et al., 2022). Due to the close resemblance in size between Cuscuta spp. and alfalfa seeds, the presence of Cuscuta spp. in the seed crop not only directly affects seed production but also presents substantial challenges for seed finishing. The dispersal of Cuscuta spp. in the alfalfa seed-growing region is another major issue. Also, a significant issue is the ability of Cuscuta spp. seeds to retain germination in the soil for decades and germinate again after alfalfa resowing (Jayasuriya et al., 2008; Olszewski et al., 2020). Especially this was a problem for seed production in the eighties of the last century (Čuturilo & Nikolić, 1986). It is necessary to study the advantages of extracting the seeds of Cuscuta sp. from natural alfalfa seeds on a laboratory machine Laboratory magnetic separator by German manufacturer Emceka Gompper and compare it with the efficiency of extracting Cuscuta sp. seeds in a conventional way. Further studies of weeds Cuscuta spp. in Serbia have given some solutions in some aspects, but the question of Cuscuta spp. is not close to being fully solved (Sarić-Krsmanović et al., 2020; 2022). It should be added that the seeds of Cuscuta spp. in alfalfa seed are considered quarantine weeds which means that such seed cannot be placed on the market Gazette of SFRY, 1987 (47/87), Gazette of the Republic of Serbia, 45 2005, and supplement to 2021 which is in line with ISTA rules (ISTA 2021), which mainly deals with quality and seed trade in Europe. The aim of this research was to extract Cuscuta spp. seeds from four seed lots of natural alfalfa seed, and examine its variability in seed size. Then, to determine whether the size of Cuscuta spp seeds affects the efficiency of seed finishing. Additionally, to contrast the effectiveness of Cuscuta spp. seed separation from natural alfalfa seeds on a laboratory magnetic separator made by German company Emceka Gompper with the effectiveness of Cuscuta spp. seed separation on equipment used in the industrial-standard process of processing natural alfalfa seeds. Materials and Methods Seeds of four seed lots of alfalfa were collected from the Banat region (the part of Serbia where alfalfa for seed production is grown the most). The seed lots were selected on the basis of a test sample, according to which the seed lots (I to IV) of natural alfalfa seed had approximately the same amount of quarantine weed Cuscuta spp. The analyzes were performed in an accredited laboratory for seed quality control, Institute of Plant Protection and Environment Belgrade. A precise electronic scale was used to measure the samples. The seed samples for analysis weighed 5 g (working) samples in accordance with the regulations of the Law on Seeds and Planting Material. All conditions related to the method of production, processing, use, circulation, import and testing are prescribed seeds of agricultural plants (Gazette of the SFRY No. 47, 1987 and Official Gazette of the Republic of Serbia No. 45, 2005). The quality of alfalfa seeds should correspond to the Regulation on the quality of seeds of agricultural plants, which is harmonized with the international regulation on the seed quality ISTA 2021. Subsample I: A sample of 5 grams of natural alfalfa seed was obtained from each seed lot. The length and width of the dodder seeds from this weed were measured using the random selection method on a sample of 30 seeds. The length and width of the seeds were examined with an Olympus BH-2 microscope. For each seed lot, the average, lowest and highest value (Min. and Max.), standard deviation (StDev), coefficient of variation (CV%), and standard error of mean were determined to determine the variation in seed size of Cuscuta spp. (SEM). For each seed lot, the correlation coefficients (r) between Cuscuta spp. seeds’ length and width were computed. Subsample IIa: From each seed lot, 1 kg of seeds was divided into four parts (repetitions) and cleaned on a laboratory magnetic separator. Subsample IIb: In the seed processing facility of the company „Herba” d.o.o. Belgrade, the seed lots are processed in the traditional industrial manner, also in four repetitions. All seed batches of natural alfalfa were processed using a selector from the CIMBRIA brand, model DELTA super 102. This selector features three shaker shoes with two 625 mm x 800 mm sieves each, for a total of six 3 m2 sieves. I shaker shoe has two sieves with round holes of 2.25 mm and 2.25 mm, II shaker shoe has two sieves with round openings of 1.4 mm and 1.4 mm, and III shaker shoe has two sieves with rectangular openings of 0.7 mm and 0.5 mm (#-wire). Sieves with the following 58 BIOLOGICA NYSSANA ● 14 (1) June 2023: 57-63 Đokić et al. ● Impact of Cuscuta spp. seed size variability on machine operation during seed finishing of natural alfalfa seeds BIOLOGICA NYSSANA ● 14 (1) June 2023: 57-63 Đokić et al. ● Impact of Cuscuta spp. seed size variability on machine operation during seed finishing of natural alfalfa seeds is the passage through the magnetic separator, where the seeds of the dodder are separated, as well as other harmful weeds and impurities that are undesirable in alfalfa seeds. Separation of weeds, especially weeds with wrinkled and unsmooth seeds and other impurities, on this machine is done by electromagnetic force. A certain quantity of steel powder with water is used for the technological process of weed separation on a magnetic separator. Between the results of the separation of Cuscuta spp. seeds, obtained by industrial and laboratory processing, the correlation coefficient (r) was calculated for each seed lot of natural alfalfa seeds. Statistical analysis was done via the freeware software package Minitab 16. Results and discussion In our experiments, dodder seed, isolated from natural seed lots, showed variability in terms of average seed size (Tab. 1). The highest average length and width of seeds of Cuscuta spp. was in the third seed lot (1,361 µm and 1,197 µm), which is 371 µm more in length, and 330 µm more in width, compared to the seed lot with the smallest seed (lot IV: length 990 µm and width 867 µm). perforations are used in practice to remove Cuscuta spp. quarantine weeds from alfalfa: 1.85 mm, 2.00 mm, and 2.25 mm. Other seeds (like Lolium sp.) are typically sieved using sieves with openings smaller than 4.5 mm. The sieves are arranged in size order from top to bottom, with larger opening sieves at the top and smaller opening sieves at the bottom. Seeds are divided into length and width using rectangular and round sieves, respectively. based on the results of the purity test of the natural seed sampled (determination of the type of inert substances, the presence of weeds and quarantine weeds Cuscuta sp., Rumex sp., Ambrosia sp. and other impurities). Depending on the level of purity of the natural seed achieved after the primary seed finishing on the CIMBRIA selector, a subsequent step of seed processing on the CIMBRIA trier model HSR 4020 RL may be required. A total of two segments, one for round seed and the other for long seed, make up the mantle, which is about 600 mm in diameter and 2,000 mm long. A mantle with alveoli with a diameter of less than 2 millimetres was employed in this instance of completing alfalfa seeds. A CIMBRIA gravity table can be used in the seed processing procedure if necessary. The last, final phase of alfalfa seed processing 59 Table 1. The seed size variability of Cuscuta spp. detected in four seed lots of natural alfalfa seed Cuscuta spp. seeds (serial number) Seed dimensions of Cuscuta spp. detected in a seed lots of natural alfalfa seeds (μm) I II III IV Length Width Length Width Length Width Length Width 1 1053 951 1275 879 1256 1156 1189 1025 2 1062 988 1283 1157 1325 1265 956 845 3 971 1109 1495 1530 1256 1253 899 802 4 1254 1086 1210 1169 1152 989 1075 895 5 1225 1114 1340 1156 1155 1156 1172 956 6 1102 978 1380 1155 1456 1356 856 802 7 1221 1121 1434 1423 1352 1302 848 832 8 1489 1178 1340 1049 1299 1255 1025 1002 9 1164 1014 1183 1127 1158 1199 975 887 10 1206 1066 1196 1073 1522 1325 887 799 11 1381 1114 1298 1181 1632 1025 926 802 12 1094 1026 1564 1346 1452 1152 1175 1011 13 1145 1128 1090 971 1289 1136 956 789 14 1178 1078 1116 1098 1325 1256 875 803 15 1301 1082 1278 1151 1256 1302 1125 1033 16 1236 1027 1198 1140 1488 1235 963 805 17 1341 1341 1313 1051 1502 1156 1023 921 18 1131 1131 1309 1155 1558 1235 985 875 Đokić et al. ● Impact of Cuscuta spp. seed size variability on machine operation during seed finishing of natural alfalfa seeds BIOLOGICA NYSSANA ● 14 (1) June 2023: 57-63 60 Cuscuta spp. seeds (serial number) Seed dimensions of Cuscuta spp. detected in a seed lots of natural alfalfa seeds (μm) I II III IV Length Width Length Width Length Width Length Width 19 1143 1143 1211 1068 1365 1099 885 756 20 1208 1208 1619 1398 1236 1156 1025 901 21 1078 1078 1201 1030 1320 1299 1125 1023 22 1347 1347 1330 1272 1398 1005 982 789 23 1214 1214 1193 971 1299 1125 976 821 24 1379 1379 1488 1084 1365 1233 1087 902 25 1242 1242 1211 1063 1455 1302 965 842 26 1314 1314 1269 1163 1266 1235 875 702 27 1347 1347 1448 1172 1566 1099 1023 825 28 1231 1231 1373 1196 1488 1263 986 756 29 1306 1021 1188 1051 1259 1198 831 799 30 1221 956 1626 1221 1368 1022 1025 1002 Average 1219 1134 1315 1150 1361 1197 990 867 Min 971 951 1090 879 1152 989 831 702 Max 1489 1379 1626 1530 1632 1356 1189 1033 StDev 115.2 123.1 140.6 138.0 127.7 99.99 100.7 92.59 CV (%) 9.447 10.86 10.69 12.00 9.383 8.356 10.17 10.68 SEM 13.97 14.93 17.05 16.74 15.48 12.13 12.21 11.23 For the dodder seed width, the highest variability was in the second seed lot (CV=12.00%), and the lowest was in the third seed lot (CV=8.356%). The variability of seed size that occurred in different seed lots can be statistically considered low (Hadživuković, 1991). Ho & Costea (2018) stated significantly higher variability for the seed size of Cuscuta spp. However, this study was done at different geographical distances and was different from the region where our experiment had been placed. Within the genus Convolvulaceae, the seed size of Cuscuta spp. is most affected by the species (Costea et al., 2015a,b). The following physical properties are considered to be the most important for seed finishing: humidity, shape, dimensions, sphericity, mass of 1,000 seeds, volume and porosity, volume-hectolitre mass, density, static and dynamic angle of internal friction (free fall angle), and external condition seed surfaces (Copeland & McDonald, 2004; Black et al., 2006; Babić & Babić, 2012; Baskakov et al., 2018). In our trials, all seed lots showed a positive correlation between seed length and width, whereas three seed lots (I, II and IV) had a very high correlation between these features (p≤0.001). In seed lot III, seed length and width did not correlate significantly (p≥0.05). Also, positive correlative interdependence was high in all seed lots (p≤0.001) (Tab. 2). Healthy alfalfa seeds have a smooth surface, and after mixing the seeds with steel powder and water in a certain proportion, the steel powder does not stay on that surface. In contrast, the dodder seed has a grain that is porous, and steel powder remains on it, which allows it to separate from alfalfa seeds (Fig. 1, Fig. 2). Table 2. Simple correlations (r) between length and width of Cuscuta spp. seeds (n=30) Seed dimensions of Cuscuta spp. originating from lots of natural alfalfa seed (μm) I II III IV Length Width Length Width Length Width Length Width r=0.579*** r=0.672*** r=0.046 ns r=0.818*** * p≤0.05, ** p≤0.01, *** p≤0.001, ns – not significant p≥0.05 Đokić et al. ● Impact of Cuscuta spp. seed size variability on machine operation during seed finishing of natural alfalfa seeds The electromagnetic cleaning machine is used for the final cleaning of alfalfa seeds from the seed of weeds. The quality of weed cleaning on this machine depends on the correct ratio of water and magnetic seed cleaning powder and the amount of seed passed through the magnetized cleaning rollers (Đokić & Stanisavljević, 2012; Uhlarik et al., 2018; Đokić et al., 2020). In our experiment, a simple correlation coefficient between the length and width of Cuscuta spp. seeds Fig. 1. Laboratory magnetic separator German man- ufacturer Emceka Gompper was very significant (r=0.784; Tab. 3). BIOLOGICA NYSSANA ● 14 (1) June 2023: 57-63 61 Fig. 2. Seed of Cuscuta spp. Table 3. Simple correlations (r) between length and width of Cuscuta spp. seeds separated from all seed lots of natural seed (n=120) Length Width r=0.784*** ***, p≤0.001 The principle of operation of electromagnetic separators (trifolin machine-detector) is based on electromagnetic action. They are intended for separating weed seeds with wrinkled and unsmooth surfaces and other impurities. The success of removing the dodder from alfalfa seeds also depends on the method of applying the appropriate metal powder (Milošević et al., 1996). Electromagnetic seed cleaning machines are very important due to their high-quality cleaning performance. Such a high quality of cleanliness cannot be achieved by pneumatic cleaning, triers or selector screens (Kozlov, 2013.). The seed of Cuscuta spp. was completely isolated from all seed lots of alfalfa seeds in our laboratory tests. Also, the seed size did not affect the separation efficiency (Tab. 4). Table 4. Efficiency (%) of separating Cuscuta spp. seeds on a laboratory magnetic separator Seed size Cuscuta spp. (length - D μm and width W μm) separated from seed lots of natural seed of alfalfa I II III IV L W L W L W L W 100a 100A 100a 100A 100a 100A 100a 100A a - Tukey’s test (p≤0.05) for length, A - Tukey’s test (p≤0.0) for width In seed finishing practice, the assumption is that laboratory and industrial processing of alfalfa seeds should be equally or similarly effective in removing Cuscuta spp. from natural alfalfa seeds. Table 5. Simple correlations (r) between the isolation of Cuscuta spp. from four seed lots of natural alfalfa seeds in laboratory conditions and in conventional alfalfa seed processing (n=4). Seed lots of natural alfalfa seed I II III IV r=0.984** r=0.931* r=0.999*** r=0.966** * p≤0.05, ** p≤0.01, *** p≤0.001, ns – not significant (p≥0.05) 62 BIOLOGICA NYSSANA ● 14 (1) June 2023: 57-63 In our study, there was a positive and highly cor- related relationship (p≤0.05 to p≤0.001) between the efficiency of magnetic separators used in the labora- tory and those used in industry (Tab. 5), which is in agreement with the results of Lin et al. (2022). Conclusion An average sample of thirty seeds of Cuscuta spp. taken from four seed lots, differed by 371 μm in length and by 330 μm in width. Dodder seed isolated from all four seed lots showed variability for width from CV=8.356 (seed from the third lot) to CV=12.00% (seed from the second lot). Between the length of the seeds of Cuscuta spp. and width was a positive correlation, but at a different level of statistical significance (p≥0.05 to p≤0.001). The influence of Cuscuta spp. seed size in the process of its separating from natural alfalfa seeds was not significant. There was a positive and significant correlation (p≤0.05 to p≤0.001) between the separation of Cuscuta spp. seeds by laboratory and industrial magnetic separator. According to the law on seed material, processed alfalfa seeds must not contain any seeds of quarantined weed Cuscuta spp, regardless of the type and dimensions of Cuscuta spp. With electromagnetic cleaning machines, the seeds of the Cuscuta spp. must be completely removed from the alfalfa seeds that are being processed. Acknowledgements. Research was financed by the Ministry of Education, Science and Technological Development, Republic of Serbia (Contract reg. No. 451- 03-68/2022-14/200383, 451-03-68/2022-14/ 200010, 451-03-68/2022-14, 451-03-9/2022-14/200133) References Abdel-Khalik, K.N. (2006). Seed morphology of Cuscuta L. (Convolvulaceae) in Egypt and its systematic significance. 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