Bioscience Journal | 2022 | vol. 38, e38074 | ISSN 1981-3163 1 Wuye Ria ANDAYANIE1 , Martin LUKITO1 , Netty ERMAWATI2 1 Department of Agrotechnology, Merdeka Madiun University Madiun 63133, Indonesia. 2 Department of Agricultural and Production, State of Polytechnic of Jember, Jember 68120, Indonesia. Corresponding author: Wuye Ria Andayanie wuye.andayanie@gmail.com How to cite: ANDAYANIE, W.R., LUKITO, M. and ERMAWATI, N. Combined effect of corn in the barrier crop and plant extracts against Cowpea mild mottle virus infecting soybean [Glycine max (l.) merr.] in the field. Bioscience Journal. 2022, 38, e38074. https://doi.org/10.14393/BJ- v38n0a2022-59636 Abstract Cowpea mild mottle virus (CpMMV) is one of the problems that can decrease soybean production. The research was conducted on the combined effects of corn in the barrier crop with plant extracts against CpMMV infecting soybean in the field. The field data was conducted using a Completely Randomized Design. The mean of disease incidence and disease severity is measured from total plants in each replicate plot on each treatment. Planting one or two of corn lines were grown at the edge four weeks before planting soybeans. Cashew nut shell (CNS), pagoda leaf, and rhizome of ginger extracts were applied using the sprayer and applied at 24 h before virus acquisition and transmission by whiteflies. The result showed that the virus incubation period ranged from 9−38 days after transmission longer than the untreated control. Planting two corn lines at the edge with CNS extract as bioactivator on soybean was the most extended incubation period of the virus and the lowest absorbance value DAS-ELISA of 0.20. There was a 73.11 % increase in the relative inhibition level of the virus. Planting corn at the edge with CNS extract proved to be more effective than soybean monoculture with CNS extract. However, soybean monoculture with CNS extract provides a better relative inhibition level of the virus (64.32 %) than planting two rows of corn on the edge combined with ginger of rhizome extract and planting two rows of corn on the edge with pagoda leaf extract as bioactivator on a soybean plant. Keywords: Bemisia tabaci. Bioactivators. Tolerance Inductor. Windbreaks. 1. Introduction The Soybean [Glycine max (L.) Merr.] is the essential agriculture commodity as the major source of protein. Cowpea mild mottle virus (CpMMV) is one of the problems that can decrease soybean production. Whiteflies (Bemisia tabaci) are known as vectors of CpMMV and spread from plant to plant by wind. They transmitted CpMMV manner, causing severe (CpMMV-S) and mild (CpMMV-M) disease symptoms (Andayanie et al. 2019). The virus can be transmitted either by seed or mechanical inoculation. The viruliferous population of whitefly in the tropical areas turned out to be the outbreak of CpMMV. The CpMMV is widely distributed in Indonesia and cause a variety of symptoms on soybean such as mild chlorotic blotches, green mosaic, distortion, blistering, and stunting. The number of resistant varieties and virus-free seeds is not sufficient for farmers' needs. Control strategies are used to anticipate more significant losses due to whitefly infestation. Management of CpMMV usually controls its vector using insecticide, which harms the environment, non -target insects, and COMBINED EFFECT OF CORN IN THE BARRIER CROP AND PLANT EXTRACTS AGAINST Cowpea mild mottle virus INFECTING SOYBEAN [Glycine max (L.) Merr.] IN THE FIELD https://orcid.org/0000-0002-5678-0742 https://orcid.org/0000-0002-8129-9053 https://orcid.org/0000-0003-0698-7507 Bioscience Journal | 2022 | vol. 38, e38074 | https://doi.org/10.14393/BJ-v38n0a2022-59636 2 Combined effect of corn in the barrier crop and plant extracts against Cowpea mild mottle virus infecting soybean [Glycine max (l.) merr.] the fie phytotoxicity. Bioactivators of ginger rhizome, cashew nut shell, and neem leaf extracts helped decrease the disease incidence and severity, symptoms, and virus concentration, respectively in soybean plants. Moreover, the whitefly nymphs of different stages (first to the third instar) could be suppressed by cashew nut shell extracts (Andayanie and Ermawati 2019). Barrier crops are used to windbreaks around the main crop and considered decreasing the ability of virus infections transmitted non-persistent because the total number of viruliferous aphids landing on the protected crop is significantly reduced (Boiteau et al. 2009; Hooks and Fereres 2006). Consequently, barrier crop may be potential alternatives aphid landing before taking off to host for the virus and the vector. Insect vectors are diverted to eat the main crops and control several viruses such as the Potato leaf roll virus (PLRV), Potato virus Y (PVY), and Bean yellow mosaic virus (BYMV) (Jones 2005; Gobiye et al. 2016). Barrier crop as corn (Zea mays L.) was also effective against Pepper veinal mottle virus (PVMV) by planting of corn on the edge, and intercrops (Kapoor 2012). Barrier crops should decrease the ability of nonpersistently aphid to transmit viruses to the primary crop. However, the corn potential for the successful use of barrier plants as vector insect management tactics still receives less attention than other management strategies. This is attributable to the fact that commercial soybean farmers commonly used insecticides such as Imidacloprid. El-Sawy et al. (2017) and Aderiye et al. (2015) noted that plant extract’s bioactive compound reduces inoculum resources of viral diseases. The bioactivator could play a key role in systemic resistance against plant viruses. This was also supported by Andayanie et al. (2019) who developed Anacardium occidentale Linn, Azadirachta indica A. Juss. and Zingiber officinale Rosc. as a source of tolerance inductor for CpMMV infection on a soybean plant. However, the use of tolerance inductor to CpMMV is still limited to a screen house experiment. Therefore, this study’s objectives were to reduce the ability of whitefly-transmitted diseases and suppress CpMMV infection that leads to sustainable and environmentally friendly by barrier crop as windbreaks and plant extract as bioactivator on soybean in the fields. 2. Material and Methods The experiment was conducted at Merdeka Madiun University, Indonesia - Department of Agrotechnology, Plant Protection Laboratory. The field experiment was initiated in March 2020 on alluvial soil at the regional Research Center (21°29'S and longitude 31°50'E) in the village Jenggrik, District Kedunggalar, Ngawi, Indonesia. Propagation of Cowpea mild mottle virus The CpMMV isolates were obtained from a collection of the Plant Protection Laboratory. The inoculum was propagated in the soybean Wilis variety. The leaf surface of soybeans seven days after planting was dusted with carborundum and smeared. The CpMMV isolate was inoculated by smeared and maintained in the greenhouse at 29 °C, with 12 h dark, 12 h light period as a source of inoculum. Leaf mosaic and rugose mosaic symptoms appeared during the eight days after inoculation and were used as an inoculum source. Preparation of plant extracts Shells were collected from waste product of cashew nut (Anacardium occidentale L.) processing in Wonogiri-Indonesia. Pagoda leaf (Clerodendrum japonicum Thunb.) and rhizome ginger (Zingiber officinale Ros.) were harvested at 90 days after planting (DAP). The different parts of plants include cashew nutshell, pagoda leaf and rhizome ginger washed in tap water and rinsed with distilled water for drying. The dried samples of cashew nutshell were ground to fine powdered form. Extraction of powdered cashew nutshell was carried out by maceration with n-Hexane in a ratio of 1:10 (w/v). The extract was filtered and evaporated with a rotary vacuum evaporator at a temperature of 55−60 oC at low pressure (450−500 mm Hg). Filter was re-macerated with chloroform solvent and re-evaporated under low pressure. The viscous extract was mixed with distilled water in a ratio of 0.3:10 and added 0.5 mg Tween 80 with stirring (Andayanie et al 2018; Andayanie et al 2019). The extractions of pagoda leaf (Clerodendrum japonicum Thunb.) and ginger (Zingiber officinale Ros.) rhizome were carried out by maceration with distilled water in a ratio of 1:10 (w/v). Cashew Bioscience Journal | 2022 | vol. 38, e38074 | https://doi.org/10.14393/BJ-v38n0a2022-59636 3 ANDAYANIE, W.R., LUKITO, M. and ERMAWATI, N. nutshell, pagoda leaf, and rhizome of ginger are used as bioactivators against CpMMV in the soybean plant. The plant extract was applied using the sprayer and applied at 24 h before virus acquisition and transmission by Bemisia tabaci. Preparation of viruliferous aphids and transmission of CpMMV Bemisia tabaci from the field was released from taro leaves overnight in the cage net. After that, a large colony of the first instar was kept on healthy soybean plants until a winged populat ion appears. The Bemisia tabaci of second instar nymphs were removed to soybean plants infected with CpMMV. The plants were covered with the cage net. Transmission of the virus is carried out by releasing Bemisia tabaci adults that have eaten the acquisition of soybean plants infected with CpMMV at four corner points of the experimental field. Observation of variables The observational variables were observed as follows: 1. Incubation periods and characteristics symptom. The incubation period was calculated from the first symptoms of soybean-based on visual observation due to the treatment. 2. Disease incidence carried out since the first time the symptoms appeared to the sixth week at intervals once a week. The disease incidence is measured from the total main plants in each replicate plot on each treatment. It was calculated using the formula: DI = a x 100% N Where, DI : disease incidence (%) a : numbers of the plant with CpMMV symptoms per plot N : numbers of the plant observed per plot 3. Disease severity is measured from total main plants in each replicate plot on each treatment. It was calculated from the first week to the sixth week after CpMMV infected and based on a scale of 0-5 as described early by Andayanie et al. (2019); 1 = plants healthy without visible symptoms on all leaves; 2 = mild chlorotic blotches symptoms (10 to 30 % of the leaves infected) ; 3 = moderate symptom (green mosaic and distortion) (30 to 50% of leaves infected); 4 = prominent symptom with blistering, and stunting (51 to 70% of leaves infected); 5 = highly severe symptoms with stunting (> 71 %). Symptom score values were converted to disease severity scores. The percentage of disease severity was calculated by the following formula: DS = Ʃ (n x v) x 100% N x V Where, DS: the disease severity (%) n : the sum of infected leaves in each category v : value score of each category N : total number of observed leaves per plant V : the highest category 4. Disease severity data were used to calculate the area under the disease progress curve (AUDPC) as described by Campbell and Madden (1990). The AUDPC value was calculating according to the formula by Asare-Bediako et al. (2018). n AUDPC =Ʃ [y1 + yi +1] (t1+ 1− t i) i=1 2 Where, Bioscience Journal | 2022 | vol. 38, e38074 | https://doi.org/10.14393/BJ-v38n0a2022-59636 4 Combined effect of corn in the barrier crop and plant extracts against Cowpea mild mottle virus infecting soybean [Glycine max (l.) merr.] the fie AUDPC : Area under disease progress curve Yi : an assessment of disease (%) at the i th observation ti : time (in weeks) at the i th observation n : the total number of observation The percentage relative inhibition level (RIL) of disease for the test at each treatment is calculated using the formula: 1 – AUDPC treatment x 100% RIL disease = AUDPC control Where, RIL disease: The relative inhibition level of disease (%) AUDPC : Area Under Disease Progress Curve Serological CpMMV detection CpMMV titers are detected serologically in samples aged four weeks after the transmission (WAT). Double-antibody sandwich-enzyme linked immunosorbent assay (DAS) ELISA method using antibody kit CpMMV (plus positive control) with procedures according to the antiserum manufacturer's guidelines (DSMZ, Braunschweig, Germany) was performed to identify CpMMV in the main plot. Test samples were taken from 10 plant samples from each treatment plot. Each treatment was detected by composite each of six composite samples representing replications of each treatment. The test is positive if the absorbance value of ELISA (AVE) of the test plant is twice the negative control (healthy) of the absorbance value of ELISA. The percentage relative inhibition level (RIL) of virus for the test at each treatment is calculated using the formula: RIL virus = AVE control – AVE treatment x 100% AVE control Where, RIL virus = the relative inhibition level of virus (%) AVE = the absorbance value of ELISA Statistical analyses The field data was conducted using a Completely Randomized Design with ten treatments. Six plots on each treatment were used as replicates. The mean of disease incidence and disease severity are measured from total plants in each replicate plot on each treatment. The experiment was done in each plot size 2.6 x 6 m and spacing 60 cm with two seeds/per hole for soybean plant. Planting one or two corn lines were grown at the edge four weeks before planting soybeans. The data were subjected to analysis of variance (ANOVA) using the statistical program of SPSS. Treatment means the difference was determined by Duncan’s Multiple Range Test (DMRT) at P< 0.05. 3. Results The symptoms varied from healthy, presumably healthy, mild mottling, mild chlorotic blotches, moderate mosaic. Mosaic, rugose mosaic, and stunting symptoms were seen on the untreated control. The incubation period of treatment plants ranged from 9−38 days after transmission longer than the untreated control. There was no significant difference (p < 0.05) with the untreated control on the incubation period of CpMMV (Table 1). The effect of planting corn lines at the edge and plant extract as bioactivator treatments in disease incidence was observed at the 1st to 7th weeks after the transmission (WAT) of Bemisia tabaci. These results suggest that planting corn lines at the edge with plant extract as bioactivator on soybean significantly reduced the disease incidence. This study showed that corn border crop with plant extract had a significant difference in the disease incidence from 3rd to 7th WAT. The control treatment had the highest CpMMV Bioscience Journal | 2022 | vol. 38, e38074 | https://doi.org/10.14393/BJ-v38n0a2022-59636 5 ANDAYANIE, W.R., LUKITO, M. and ERMAWATI, N. disease incidence at the seventh WAT. There was no significant difference (p < 0.05) with soybean monoculture plus pagoda leaf extract as bioactivator on soybean (SMC + PL) from 6th to 7th WAT. Planting two corn lines at the edgewith cashew nutshell extract as bioactivator on soybean (BC 2 R + CNS) recorded the lowest disease incidence during the 6th and 7th WAT. During the 6th and 7th WAT, the stability of the disease incidence was noted on BC 2 R + CNS. There was no significant difference in disease incidence for the difference corn densities at both weeks’ recordings (Table 2). Table 1. Effect of treatments on the incubation period and typical symptom. Treatments Incubation periods (day) Typical symptoms BC 1 R + CNS 25.87 ± 2.33b Healthy BC 2 R + CNS 38.61 ± 2.17a Healthy SMC + CNS 23.49 ± 2.72b Healthy BC 1 R + PL 12.70 ± 3.41e Mild chlorotic blotches BC 2 R + PL 16.20 ± 2.70d Healthy, presumably healthy, early signs of senescing SMC + PL 9.85 ± 1.65f Mild chlorotic blotches, moderate mosaic BC 1 R + RG 19.55 ± 2.69c Healthy BC 2 R + RG 21.68 ± 2.76c Healthy, early signs of senescing SMC + RG 16.47 ± 3.19d Presumably healthy, mild chlorotic blotches Control 8.11 ± 1.76f Mosaic, rugose mosaic, and stunting Note: BC 1 R + CNS= planting one corn line at the edge with cashew nut shell extract as bioactivator on soybean; BC 2 R + CNS= planting two corn lines at the edge with cashew nut shell extract as bioactivator on soybean; SMC + CNS = soybean monoculture with cashew nut shell extract as bioactivator on soybean; BC 1 R + PL= planting one corn lineat the edge with pagoda leaf extract as bioactivator on soybean; BC 2 R + PL= planting two corn lines the edge with pagoda leaf extract as bioactivator on soybean; SMC + PL = soybean monoculture with pagoda leaf extract as bioactivator on soybean; BC 1 R + RG= planting one corn lines at the edge with rhizome of ginger extract as bioactivator on soybean; BC 2 R + RG= planting two corn lines at the edge with rhizome of ginger extract as bioactivator on soybean; SMC + RG = soybean monoculture with rhizome of ginger extract as bioactivator on soybean; Control = soybean monoculture without plant extract as bioactivator on soybean. According to the Duncan Multiple Range Test, values sharing the same letters differ non significantly (P < 0.05). Table 2. The development of disease incidence at weeks after the transmission. Treatments Incidence of Cowpea mild mottle virus from ... WAT), % 1 2 3 4 5 6 7 BC 1 R + CNS 0.00b 0.00c 2.88d 3.14e 4.12f 5.78e 6.94e BC 2 R + CNS 0.00b 0.00c 2.64d 2.91e 3.98f 4.15e 4.15e SMC + CNS 0.00b 2.10c 6.42c 9.55d 12.76e 16.20d 18.25d BC 1 R + PL 0.00b 9.83ab 18.26bc 22.80b 32.95c 45.85b 59.12b BC 2 R + PL 0.00b 4.92 bc 15.54bc 16.12c 35.26c 36.52c 42.87 bc SMC + PL 0.00b 11.56ab 24.70ab 33.75a 47.09b 53.17a 70.11a BC 1 R + RG 0.00b 5.27bc 10.73c 20.60b 24.39d 33.08c 36.62c BC 2 R + RG 0.00b 2.98c 8.85c 11.82c 15.64e 18.45d 21.12d SMC + RG 0.00b 6.75b 17.07b 24.96b 26.78d 47.14b 58.66b Control 9.31a 17.48a 31.05a 38.23a 54.71a 56.82a 73.29a Note: WAT = weeks after the transmission. According to the Duncan Multiple Range Test, values sharing the same letters differ non significantly (P < 0.05). Planting two corn lines at the edge with extract of cashew nutshell as bioactivator on soybean (BC 2 R + CNS) recorded the lowest disease severity during the 3rd to the 7th WAT. It was shown a similar pattern with planting one corn line at the edge with extract of cashew nutshell on soybean (BC 1 R + CNS) on disease severity during the 1st to the 7th WAT. There were significant differences when compared to soybean monoculture with CNS extract (SMC + CNS) at the 6th and the 7th WAT. During the 6th and the 7th WAT, the worst performance was planting soybean monoculture with pagoda leaf extract. It showed no significant difference from the control treatment. However, planting two corn lines at the edge with pagoda leaf extract as bioactivator on soybean (BC 2 R + PL) and planting two corn lines at the edge with rhizome of ginger extract as bioactivator on soybean (BC 2 R + RG) performed better than one corn line with pagoda leaf extract as bioactivator on soybean (BC 1 R + PL) and planting one corn line at the edge with rhizome of ginger extract as bioactivator on soybean (BC 1 R + RG), respectively (Table 3). The analysis at the 4th WAT of data in Table 4 revealed that planting two corn lines at the edge with cashew nut shell extract as bioactivator on soybean (BC2R + CNS) had the lowest area under the disease progress curve (AUDPC) of 02.72. There was no significant difference (P<0.05) from planting one corn at the Bioscience Journal | 2022 | vol. 38, e38074 | https://doi.org/10.14393/BJ-v38n0a2022-59636 6 Combined effect of corn in the barrier crop and plant extracts against Cowpea mild mottle virus infecting soybean [Glycine max (l.) merr.] the fie edge with cashew nutshell extract as bioactivator on soybean (BC1R + CNS) but was significantly different (P<0.05) from the remaining eight treatments. Planting two corn lines at the edge with cashew nutshell extract as bioactivator on soybean (BC 2 R + CNS) had the highest relative inhibition level (RIL) of disease (73.11 %) and RIL of virus (61.79 %). There was the lowest absorbance value of ELISA (AVE). The titer of CPMMV concentration reached 0.20 while the AVE of the negative sample was 0.28 (the data is not shown in Table 4). The treatments BC 2 R + PL, BC 1 R + RG, SMC + RG provided no significant differences (p < 0.05). Among test treatments, the absorbance value test sample of control, planting one corn line at the edge with pagoda leaf extract as bioactivator on soybean (BC 1 R + PL) and soybean monoculture with pagoda leaf extract as bioactivator (SMC + PL) showed positive reaction with absorbance value of two times larger than healthy control plants. Table 3. The development of disease severity at weeks after the transmission. Treatments Disease severity of Cowpea mild mottle virus from ... WAT), % 1 2 3 4 5 6 7 BC 1 R + CNS 0.00b 0.00d 0.83f 1.19e 2.26d 4.86e 4.86f BC 2 R + CNS 0.00b 0.00d 0.00f 0.71e 0.74d 1.22e 1.24f SMC + CNS 0.00b 0.03d 2.25ef 3.40e 1.57d 10.78d 12.48e BC 1 R + PL 0.00b 3.19c 14.48c 19.33c 31.65b 44.65b 53.29b BC 2 R + PL 0.00b 2.88c 10.15d 10.88d 20.11c 28.78c 36.60c SMC + PL 0.00b 4.72b 18.94b 27.12b 38.45b 53.01a 71.47a BC 1 R + RG 0.00b 2.66c 9.22d 12.18d 11.02cd 25.21c 29.98d BC 2 R + RG 0.00b 2.15c 4.78e 4.78e 9.26c 14.62d 14.99e SMC + RG 0.00b 3.94b 9.51d 10.28d 14.75cd 31.10c 38.05c Control 3.66a 9.01a 26.42a 40.90a 51.69a 58.34a 77.81a Note: WAT = weeks after the transmission. According to the Duncan Multiple Range Test, values sharing the same letters differ non significantly (P < 0.05). Table 4. Effect treatments on AUDPC, absorbance value of ELISA, disease, and virus inhibition at fourth weeks after the transmission. Treatments AUDPC RIL of disease (%) Absorbance value of ELISA RIL of virus (%) BC 1 R + CNS 04.33g 56.48 0.24e 68.03 BC 2 R + CNS 02.72g 61.79 0.20e 73.11 SMC + CNS 13.85f 54.95 0.28de 64.32 BC 1 R + PL 35.21c 27.68 0.65b 29.41 BC 2 R + PL 26.04d 34.42 0.51c 31.93 SMC + PL 41.93b 19.60 0.70a 24.82 BC 1 R + RG 20.38e 48.27 0.48c 51.03 BC 2 R + RG 15.97f 51.92 0.36d 59.70 SMC + RG 24.50d 40.83 0.59b 35.95 Control 46.15a 00.00 0.74a 0.00 Note: AUDPC = Area Under the Disease Progress Curve; RIL = Relative Inhibition Level; ELISA = Enzyme Linked Immunosorbent Assay According to the Duncan Multiple Range Test, values sharing the same letters differ non significantly (P < 0.05). 4. Discussion Planting two corn lines at the edge with cashew nutshell extract as bioactivator on soybean proved to be more effective because of the ability bioactive compound on CNS in reducing CPMMV symptoms. Moreover, the use of two corn lines at the edge can prevent the spread of B. tabaci to the main crop by the wind. This is in agreement with earlier findings by Andayanie et al. 2019 that the bioactive component in cashew nutshell extract consisted mainly of anacardic acid (6-pentadecylsalicylic acid) of 76.93%. Anacardic acid is a derivative of salicylic acid. The bioactive compound was assumed to play a role in increasing resistance to CPMMV infection. Other studies carried out by Andayanie and Ermawati (2019) have shown that the first to the second nymphal instar population density of B. tabaci could be reduced until approximately 90 % after using CNS extract at a concentration of 2.00 % in a screen house. The same trend has been observed with Aphis gossypii that a high-density border is associated with low population aphids in cotton crops (Gobiye et al. 2016). Similarly, in a study conducted by Damicone et al. (2007), no border crop could be associated with increasing aphid landing on plots. Thus, the main crop will have an increased Bioscience Journal | 2022 | vol. 38, e38074 | https://doi.org/10.14393/BJ-v38n0a2022-59636 7 ANDAYANIE, W.R., LUKITO, M. and ERMAWATI, N. level of virus symptoms. Planting one or two corn lines with CNS extract seems to affect the incidence of viral disease. There was an ability to attract B. tabaci that will prevent the migration of aphid to the main crop. Systemic acquired resistance against plant viruses can also be enhanced by natural elicitors such as bioactivator. From this study, it is evident that planting one or two corn lines with CNS extract proved to be more effective in the incidence of virl diseases. The same trend has been observed with B. tabaci on soybean plants where CNS extract had an antifeedant effect (Andayanie & Ermawati, 2019). On the other hand, Hooks and Fereres (2006) noted that the mechanism of suppression of a viral disease incidence could be expressed as follows: (1) barrier plants acted as natural sinks for non-persistent virus vectors that lose their ability to transmit viruses after the acquisition because of these viruses are lost on barrier plants (2) barrier plants may act as a physical barrier to transmit the virus if plants were taller than the main crop, and (3) barrier plants acted as camouflage or mask of host plants against the insect. Moreover, cashew nutshell, Zingiber officinale, and Clerodendrum inerme extracts significantly induced systemic resistance against CpMMV, BCMMV, Tomato yellow leaf curl virus (TYLCV), respectively compared to untreated plants (El-Sawy et al. 2017). The control plot showed the highest percentage of disease severity from the first WAT onwards. From this study, it is evident that corn as a barrier and plant extract as bioactivator on soybean proved to be more effective than non-treated control plot because of its ability to attract aphid on the barrier crop and antifeedant effect of plant extracts. It may be due to the high population of Bemisia tabaci in the control treatment. On the other hand, disease severity increases progressively without plant extract as bioctivator on soybean. Moreover, several bioactive compounds in cashew nut shells as bioactivator could play a key role in this respect. The same phenomenon was reported by Andayanie and Ermawati (2019) that cashew nutshell compounds act as antifeedant on the nymphal stage of B. tabaci. The previous study was carried out using cashew nutshell extract to cause the antifeedant effect of B. tabaci and biactivator of soybean against CpMMV. The results of Andayanie et al. (2019) also showed that cashew nutshell extract at a concentration of 2.00 % could inhibit the landing of adults Bemisia tabaci on soybean leaf. Additionally, plant extracts as bioctivator had mobilized salicylic acid content on secondary metabolite production to challenge CpMMV. It can be seen in soybean monoculture with cashew nutshell extract as bioactivator on soybean. This observation could be due to the fact that bioactive compounds in CNS extract had an inhibitory effect on landing and staying for feeding deterrence on soybean leaflets. According to the previous results obtained by Andayanie et al. (2019), the content of anacardic acid in CNS extract had the dominant role as an antifeedant active compound on B. tabaci. This implied that planting corn on the edge with pagoda leaf and rhizome of ginger extract, respectively proved to be less effective than no barrier crop with CNS extract because the bioactive compound in CNS extract was able to inhibit the landing and staying of B. tabaci to the main crop for feeding deterrence. Conversely, though, the results contradict previous findings by Hooks and Fereres (2006) who suggested that the height of barrier crop is important for non-persistent virus vector control. On the other hand, the retention period of the non-persistent virus is short of several minutes to several hours. Based on all samples, plants’ serological detection from each replicate plot none of BC 2 R + PL extract, BC 2 R + RG, and SMC + RG extract was detected to be positive for CPMMV. However, some plants showed presumably healthy and early signs of senescing symptoms at the 8th WAT. These show that can be controlled CPMMV. However, due to other pathogenic infections occurring naturally in the field, symptoms were different from those of CPMMV infection. In this study, there was no other pathogenic detection, such as fungi that infect naturally because of high soil moisture in the field. Unlike BC 1R + CNS extract, BC 2 R + CNS extract and SMC + CNS extract due to the presence of CNS extract as an antifungal. This concept distinguishes typical symptoms. This is in agreement with Garcia et al. (2018), who concluded that CNSL had the highest antifungal potential in the control of C. gloeosporioides and L. theobromae in papaya fruits C. gloeosporioides and L. theobromae caused anthracnose and fruit rot, respectively. The bioactive compound of CNSL can prevent the inhibition of mycelial growth and spore production. Similarly, in a study conducted by Andayanie et al. (2021), CNS extract had a high level of total polyphenolic, flavonoid content, pH, and a low value of titratable acidity be attributed to its potent antifungal activity. The same trend has been observed in bioactive polyphenol compounds, singly or in combination. The compounds could not stimulate fungal and viral growth. The compounds may have destroyed the cell membrane fungi (Rongai et al. 2012). Bioscience Journal | 2022 | vol. 38, e38074 | https://doi.org/10.14393/BJ-v38n0a2022-59636 8 Combined effect of corn in the barrier crop and plant extracts against Cowpea mild mottle virus infecting soybean [Glycine max (l.) merr.] the fie The use of corn on the edge with plant extracts and soybean monoculture with cashew nutshell extract can inhibit CpMMV infection and infection through B. tabaci. There was not found in the BC 1 R + PL treatment. Corn border type at high density causes the main plant target to be less recognized by B. tabaci for colonization. However, plant extracts as an impact on resistance also have a role in inhibiting CpMMV infection and disease. Other studies carried out by Elbeshehy (2017) have been used to bioactivator against plant pathogen, including bacteria, fungi, and viruses. The inducers such as CNS extract also effectively acquired soybean against CpMMV (Andayanie et al. 2019). The value of disease severity at four weeks after the transmission was directly related to AUDPC. The virus titer of CpMMV also decreased with the relative decline in symptom severity scale. Similarly, in a study conducted by Asare-Bediako et al. (2018) that the Cowpea mosaic virus of disease severity was positively related to AUDPC on cowpeas genotypes resistance. 5. Conclusions This study showed that planting corn as a barrier crop with CNS extract as bioactivator prevents migrating B. tabaci to the main crop and decreasing the relative inhibition level (RIL) of disease and CpMMV. Moreover, the use of CNS extract prevented adults B. tabaci from landing on planting soybean monoculture. There was a better decrease in RIL of disease and CpMMV than maize barrier plant with rhizome ginger extract and planting two maize barrier plant with pagoda leaf extract. Authors' Contributions: ANDAYANIE, W.R.: Conception and design, acquisition of data, analysis, and interpretation of data, drafting the manuscript, final approval; LUKITO, M: Acquisition of data, analysis, and interpretation of data, final approval; ERMAWATI, N .: Drafting the manuscript, final approval. All authors have read and approved the final version of the manuscript. Conflicts of Interest: The authors declare no conflicts of interest. Ethics Approval: Not applicable. Acknowledgments: The authors would like to thank the DRPM Ministry of Research, Technology and Higher Education of the Republic of Indonesia who has funded this research and publication through the Leading Higher. 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