Twindemic Threats of Weeds Coinfected with Tomato Yellow Leaf Curl Virus and Tomato Spotted Wilt Virus as Viral Reservoirs in Tomato Greenhouses

Nattanong Bupi; Thuy Thi Bich Vo; Muhammad Amir Qureshi; Marjia Tabassum; Hyo-jin Im; Young-Jae Chung; Jae-Gee Ryu; Chang-seok Kim; Sukchan Lee

doi:10.5423/PPJ.OA.03.2024.0049

Plant Pathol J > Volume 40(3); 2024 > Article

Bupi, Vo, Qureshi, Tabassum, Im, Chung, Ryu, Kim, and Lee: Twindemic Threats of Weeds Coinfected with Tomato Yellow Leaf Curl Virus and Tomato Spotted Wilt Virus as Viral Reservoirs in Tomato Greenhouses

Research Article

The Plant Pathology Journal 2024;40(3):310-321.

Published online: June 1, 2024

DOI: https://doi.org/10.5423/PPJ.OA.03.2024.0049

Twindemic Threats of Weeds Coinfected with Tomato Yellow Leaf Curl Virus and Tomato Spotted Wilt Virus as Viral Reservoirs in Tomato Greenhouses

Nattanong Bupi¹, Thuy Thi Bich Vo¹, Muhammad Amir Qureshi¹, Marjia Tabassum¹, Hyo-jin Im¹, Young-Jae Chung², Jae-Gee Ryu³, Chang-seok Kim^4,^*

, Sukchan Lee^1,^*

¹Department of Integrative Biotechnology, College of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon 16419, Korea

²Department of Biomedical Science, Hwasung Medi-Science University, Hwaseong 18274, Korea

³Research and Development Planning Division, Rural Development Administration, Jeonju 54875, Korea

⁴Institute for Future Environmental Ecology, Jeonju 54883, Korea

^*Co-corresponding authors: S. Lee, Phone) +82-31-290-7866, FAX) +82-31-290-7892, E-mail) cell4u@skku.edu, C.-S. Kim, FAX) +82-63-211-7123, E-mail) green8473@naver.com

Handling Editor: Ju-Yeon Yoon

(Received on March 7, 2024; Revised on April 13, 2024; Accepted on May 9, 2024)

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Tomato yellow leaf curl virus (TYLCV) and tomato spotted wilt virus (TSWV) are well-known examples of the begomovirus and orthotospovirus genera, respectively. These viruses cause significant economic damage to tomato crops worldwide. Weeds play an important role in the ongoing presence and spread of several plant viruses, such as TYLCV and TSWV, and are recognized as reservoirs for these infections. This work applies a comprehensive approach, encompassing field surveys and molecular techniques, to acquire an in-depth understanding of the interactions between viruses and their weed hosts. A total of 60 tomato samples exhibiting typical symptoms of TYLCV and TSWV were collected from a tomato greenhouse farm in Nonsan, South Korea. In addition, 130 samples of 16 different weed species in the immediate surroundings of the greenhouse were collected for viral detection. PCR and reverse transcription-PCR methodologies and specific primers for TYLCV and TSWV were used, which showed that 15 tomato samples were coinfected by both viruses. Interestingly, both viruses were also detected in perennial weeds, such as Rumex crispus, which highlights their function as viral reservoirs. Our study provides significant insights into the co-occurrence of TYLCV and TSWV in weed reservoirs, and their subsequent transmission under tomato greenhouse conditions. This project builds long-term strategies for integrated pest management to prevent and manage simultaneous virus outbreaks, known as twindemics, in agricultural systems.

Keywords: Begomovirus, orthotospovirus, reservoirs, weed management

Tomatoes (Solanum lycopersicum L.) are commonly grown in greenhouses and have become increasingly popular in South Korea because of their high nutritional value, which is attributed to the amount of nutrients and vitamins they contain such as beta-carotene and lycopene (Costa and Heuvelink, 2018; Dorais et al., 2008; Pék et al., 2010, 2011). Tomatoes are commonly consumed in two ways: as fresh fruit and for food preparation (Alda et al., 2009). The challenges faced in tomato farming extend beyond adverse weather conditions, and almost 200 tomato diseases have been identified, which continuously affect tomato production (Karienye et al., 2018; Kokalis-Burelle, 2002). Consequently, crop damage caused by plant diseases increases and productivity in manufacturing decreases, including the increasing amount of agricultural expenses, the use of chemical insecticides, and the excess application of fertilizers (Chen et al., 2014; Hanssen et al., 2010; Savary et al., 2012; Zayan, 2019). When considering tomato viral diseases, it is important to remember that tomato yellow leaf curl virus (TYLCV) and tomato spotted wilt virus (TSWV) are the two main virus-based plant diseases that commonly affect tomatoes (Ong et al., 2020). Tomatoes grown worldwide face a huge threat from TYLCV and TSWV. These viruses belong to the begomovirus and orthotospovirus genera, respectively, and are well known for causing significant economic damage to tomato crops (Dhaliwal et al., 2020; Hoang et al., 2013).

TYLCV is a member of the family Geminiviridae. It has a single-stranded circular DNA and monopartite genome (DNA-A) that is approximately 2.6-2.8 kb in size. The genome is enclosed in a twinned icosahedral structure (Czosnek and Laterrot, 1997; Ghanim et al., 1998). TYLCV is limited to the phloem tissues of its hosts and is transmitted by the whitefly (Bemisia tabaci) (Jones, 2003). Stunting, extreme leaf curling, and yellowing are the classic symptoms of tomato plants infected by TYLCV. In addition, TYLCV can spread via seeds; the virus is present widely and is rapidly disseminated to new areas, nations, and continents (Kil et al., 2016). The first occurrence of TYLCV in South Korea was recorded in 2008 (Lee et al., 2010), and since then, it has been constantly spreading throughout the country. Subsequently, over two decades ago, diseases emerged and caused severe damage to tomato crops, ultimately spreading to all regions of South Korea. In 2021, a survey of the new TYLCV variants was conducted. The findings indicated the presence of three distinct groups of TYLCV in South Korea: TYLCV-KG3, TYLCV-KG4, and TYLCV-KG5. TYLCV-KG3 and -KG4 belong to the new severe strain group, whereas TYLCV-KG5 belongs to the new mild strain group (Bupi et al., 2023). TSWV was first reported in South Korea in 2004 in sweet pepper (paprika) from Yesan (Kim et al., 2004). This virus subsequently spread to the entire nation. TSWV belongs to the Tospoviridae family (Maes et al., 2018); its genetic information includes three RNA species, namely S (2.9 kb), M (4.8 kb), and L (8.9 kb), which are negative and comprise single-stranded RNA (ssRNA) (Adkins, 2000). Their polymorphic particles have diameters ranging from 80 to 120 nm. These particles have surface projections that contain two viral glycoproteins, namely G1 and G2 (Adkins, 2000; Mahy and van Regenmortel, 2008). TSWV is transmitted by approximately 10 thrip species, including the western flower thrips (Frankliniella occidentalis) (Riley et al., 2011; Sundaraj et al., 2014). In 2019, 42 different weed species were confirmed as TSWV hosts, each with its own unique life cycle, which suggests that these weeds could serve as significant viral reservoirs in South Korea (Kil et al., 2020).

Researchers and farmers have paid close attention to TYLCV and TSWV because they are two of the most common and economically important plant viruses. The persistence and spread of these viruses remain an important hazard to tomato production despite extensive research on their epidemiology and disease management (Panno et al., 2021). In this study, weeds were hypothesized to play an important role in the epidemiology of TYLCV and TSWV. For several plant viruses, including these two viruses, weeds are known to be key hosts (Massumi et al., 2009). Furthermore, disease-carrying insects are an unknown factor in the correlation between disease occurrence and the abundance of weeds (Duffus, 1971). In this study, an attempt was made to identify the link between the factors contributing to greenhouse diseases in each period of tomato growth. The findings indicated that the perennial weed exhibited positive results for both types of viruses, including the roots of the perennial weed that are resistant to chemical treatment, and prevented farmers from practicing plant cultivation. Ongoing investigations led to additional unexpected findings: new leaves from perennial weeds collected from greenhouses showed positive detection results for both varieties of viruses. As a result, strategies that farmers should adopt to prevent the spread of diseases throughout the coming cultivation period were simplified and suggestions to attain the goal of controlling and preventing diseases in the future during the greenhouse production of crops were provided.

Materials and Methods

Sampling of tomatoes and weeds with viral diseases

In February 2023, tomato leaves showing typical TYLCV and TSWV disease symptoms were collected from Nonsan, South Korea (Fig. 1, Supplementary Fig. 1). Viral DNA/RNA was extracted from the 50 leaf samples using the Viral Gene-spin Viral DNA/RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Korea) according to the instructions provided. In addition, 130 samples of 16 weed species around tomato cultivating area inside the greenhouse were collected for viral extraction and detection. These included Bothriospermum zeylanicum, Cerastium glomeratum, Hemisteptia lyrata, Oxalis corniculata, Mazus pumilus, and Potentilla amurensis. Pseudognaphalium affine, Ranunculus sceleratus, Rorippa indica, Rorippa palustris, Rumex crispus, Senecio vulgaris, Sonchus asper, Stellaria aquatica, Trigonotis peduncularis, and Veronica peregrina. Furthermore, the selected perennial weed roots, such as C. glomeratum (n = 6), R. crispus (n = 12), R. palustris (n = 6), S. aquatica (n = 12), S. asper (n = 6), and S. vulgaris (n = 6), were collected and extracted for viral detection using the above extraction method. The percentage disease incidence (PDI) for infected tomato sample and weed sample were calculated for each of the viral symptom using the following formular (Kaushal et al., 2020).

PDI(%)=No. of infected plantsTotal no. of plants species sampling×100

Detection of TYLCV and TSWV

Viral DNA detection and polymerase chain reaction (PCR) were performed using TYLCV-specific primers, which were designed based on the sequence of the C1 coding gene previously reported in TYLCV-isolated Korea (The PCR product was 1.1 kb; forward primer: 5′-AAGCGACCAGGCGATATAATC-3′; reverse primer: 5′-AGGGGAACTCATCACTGCTC-3′). The final PCR reaction volume was 20 μl, and the reaction was performed using 1× AccuPower ProFi Taq PCR Master Mix (Bioneer, Daejeon, Korea) according to the standard protocol, as follows: initial denaturation at 95°C for 3 min, followed by 35 cycles (denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and an extension at 72°C for 1 min), and a final extension at 72°C for 10 min.

The viral RNA detection and reverse transcription (RT)-PCR were performed using TSWV-specific primers, which were designed based on the sequence of the M segment previously reported in TSWV isolated Korea (The PCR product was 1.0 kb; forward primer: 5′-TGTGTGAGTGCTTTGGATAGG-3′; reverse primer: 5′-CCGGTGGTTAGTAAACAAGAC-3′). The final RT-PCR reaction volume was 20 μl. SuPrimeScript RT-PCR Premix (GeNet Bio, Daejeon, Korea) was used, and the reaction was performed according to the manufacturer’s protocol. It included a cDNA synthesis step at 50°C for 30 min, an initial denaturation at 95°C for 3 min, followed by 35 cycles (denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and an extension at 72°C for 1 min), and a final extension at 72°C for 10 min. The PCR and RT-PCR were performed using ProFlex PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The products were electrophoresed on 1% agarose gels. In addition, the PCR results were obtained using Sanger sequencing at the Macrogen Institute (Macrogen, Seoul, Korea). The PCR and RT-PCR reactions for each DNA and RNA sample were performed a minimum of three times in this study. Three times were performed for each reaction.

Detection in the insect vectors

Samples of whiteflies (B. tabaci) (total of n = 40) and thrips (F. occidentalis) (total of n = 40) in the similar area of selected tomatoes and weeds for viral detection were collected from the Nonsan tomato farm. Viral DNA/RNA was extracted from the collected insect (n = 5 per reaction) samples using the Viral Gene-spin Viral DNA/RNA Extraction Kit according to the instructions provided. Viral DNA/RNA was detected as mentioned above for the detection of TYLCV and TSWV. It was repeated three times for each reaction.

Detection of viruses in the planted weed leaves

The perennial weed root of R. crispus was selected and planted in a controlled-environment room. After 4 weeks of planting, nucleic acid was extracted from the newly emerged apical leaves of R. crispus using the Viral Gene-spin Viral DNA/RNA Extraction Kit according to manufacturer’s guidelines and used in the analysis of TYLCV and TSWV detection as previously described. A total of three times were performed for each reaction. Datasets of detection results were arranged into class intervals for statistical range analysis using the modified formular to determine the spread of detection results (Bonassi et al., 2011).

Results

Detection and identification of viruses in collected tomato samples

To confirm the occurrence of TYLCV and TSWV via PCR or RT-PCR amplification using TYLCV- or TSWV-specific primers, viral DNA or RNA present in tomato samples was extracted and detected (Fig. 1A-D). The results of viral detection from 50 tomato samples indicated that 15 of them only showed a positive TYLCV band, exhibiting an amplicon of approximately 1.1 kb. Another 15 only showed a positive TSWV band, with an amplicon of approximately 1.0 kb. Fifteen of them showed both positive TYLCV and positive TSWV bands on gel analysis (Table 1). The PDI was 30%, 30%, and 30% on the TYLCV, TSWV, and mixed infected samples, respectively. The TYLCV PCR amplicons were sequenced, and subsequent sequence analysis of nucleotides using BLAST revealed a nucleotide sequence similarity of 97.23% with the TYLCV-isolated TK2-1, South Korea in the coat protein area (GenBank accession no. PP179273). The TSWV RT-PCR amplicons were sequenced. The resulting nucleotide sequences were analyzed using BLAST. The results showed a nucleotide sequence similarity of 98.68% with segment M of TSWV-isolated NS-AG28 (GenBank accession no. MT842840) (Supplementary Fig. 2).

Detection of TYLCV and TSWV in weed samples

To distinguish the growth cycles and propagation of the collected weeds (Rao, 2000). Five perennial, and 11 annual weeds for four growing seasons (winter, spring, fall, and summer) were collected and summarized (Table 2). To determine the connecting factors among the variables that cause disease near the tomato cultivation area, 130 samples of 16 weed species from the tomato greenhouse were subjected to PCR or RT-PCR amplification and gel analysis. Primers specific to TYLCV and TSWV were used for this purpose. The results indicated that six weed species, namely, C. glomeratum, R. crispus, R. palustris, S. aquatica, S. asper, and S. vulgaris, exhibited positive for both viral detection (Table 2). Of these, six weed species were infected by TYLCV and TSWV (Fig. 2). Furthermore, the root of six perennial weed species that showed positive results in the weed leaves viral detection were examined using the methods of TYLCV or TSWV detection. The results of root detection indicated that six weed species were positive for both viruses (Table 3, Fig. 3, Supplementary Fig. 3).

Detection of viruses in the insect vector

To confirm that TYLCV and TSWV were transmitted by insect vectors and spread widely in greenhouse conditions, insect vectors were collected via purposive sampling, and whiteflies (B. tabaci) and thrips (F. occidentalis) were focused on. The results of TYLCV and TSWV obtained via PCR or RT-PCR amplification using TYLCV- or TSWV-specific primers on gel analysis showed that all whitefly (total of n = 40) samples clearly exhibited a positive band for TYLCV, an amplicon of approximately 1.1 kb, but a TSWV-positive band did not appear on the gel analysis result. Thrips (total of n = 40) samples exhibiting a positive TSWV band, showing an amplicon of approximately 1.0 kb, but a TYLCV-positive band did not appear on the gel analysis result (Table 4, Supplementary Fig. 4). The findings from insect vector detection showed that there was no evidence of viral nucleotides in nonspecific hosts for whiteflies infected with TYLCV and thrips infected with TSWV transmitted from one to the other.

Detection in the new leaves of perennial weeds

To identify the contributing disease factors across seasons in the greenhouse and being the source of inoculation by insect vectors before the new tomato planting season. Perennial weed roots of R. crispus were planted in the plant chamber room to detect TYLCV and TSWV. After 4 week of planting, three sets (n = 9 per set) of newly emerged apical leaves of R. crispus (total of sample = 27) were analyzed (Fig. 4). The results of PCR or RT-PCR amplification indicated that seven new apical leaf showed positive results for the TYLCV PCR band and one new apical leaf demonstrated positive results for the TSWV RT-PCR band on gel analysis (Supplementary Fig. 5). Table 4 showed the statistical range analysis of TYLCV and TSWV detection results in newly produced apical leaves of R. crispus. The frequency of detection exhibiting 3 positive samples spread from approximately ‘10 to 18’ and ‘19 to 27’ from TYLCV detection results, producing a range of 16.57 new apical leaves. The frequency of detection exhibiting 1 positive sample spread from approximately ‘19 to 27’ from TSWV detection results, producing a range of 23 new apical leaves. The statistical range results indicated the possibility of R. crispus could be a green bridge of two viruses for new planting season. Based on this analysis the approximate number of new infected plants should be more than 16 and 23 on TYLCV and TSWV in greenhouse, respectively (Table 5).

Discussion

The term “twindemic” has gained popularity during the outbreaks of coronavirus disease 2019 and influenza pandemics (Ferdinand et al., 2020). The previously mentioned research also considers these viruses as causing important diseases in humans and leading to health catastrophes from 2020 onward. Similar to the agricultural system, a twindemic of TYLCV and TSWV can occur under greenhouse cultivation conditions. Both viruses exert key effects on tomato production, either directly or indirectly. Currently, there is no possibility of developing a vaccine or identifying an effective cure for plant viral diseases (Abdelkhalek and Hafez, 2020; Lal et al., 2015). These viruses have insect vectors (whiteflies and thrips) that are difficult to eradicate, and their host plants are very similar to our result, which showed the co-infection of TYLCV and TSWV in tomatoes as well as in R. crispus, S. vulgaris, and C. glomeratum.

It is significant to highlight that TYLCV and tomato chlorosis virus (ToCV), transmitted by the same vector (whiteflies), could potentially result in a concurrent outbreak in tomatoes. This is particularly noteworthy considering recent reports indicating that the presence of ToCV in tomato farms is as prevalent as that of TYLCV in South Korea (Kwon et al., 2022). The additional results of RT-PCR were analysis to test whether ToCV was mixed-infection with those tomato and insect vector samples by using ToCV-specific primers (Li et al., 2021). Thus, there were no instances of co-infection observed in the collected samples (Supplementary Fig. 6). Unexpectedly, new leaves from perennial weed roots (R. crispus) also tested positive for both viruses. R. crispus is natural hosts of whiteflies and thrips (Groves et al., 2002; Lee et al., 2008), Rumex spp. showed a germination rate of 63-90% during a period of 2-7 years (Palai, 2010; Polston et al., 2009). It is necessary to ensure that the roots of perennial weeds are removed completely during the agricultural cultivation process by translocating systemic herbicides such as glyphosate and auxinic herbicides, which could benefit perennial weed control (Davies and Peoples, 2003; Hess, 2018; Steinmann et al., 2012). We propose that the roots of perennial weeds could act as an intermediary linking the source of inoculum and the insect vector as a viral reservoir, thereby promoting the spread of the disease to new-season crops.

Fig. 5 shows that the presence of the two viruses (TSWV and TYLCV) in weed reservoirs could contribute to the spread of the virus from one season to the other under tomato greenhouse conditions, which is summarized in the following paragraphs. The insect vector that carries TSWV or TYLCV originates outside the greenhouse. Subsequently, these vectors begin to feed on young tomatoes during planting seasons, thus becoming transmitters of viral diseases (TSWV and TYLCV) over growing seasons. Insect vectors initiate feeding on perennial weeds in accordance with the harvesting season, thus initiating the accumulation of TYLCV and/or TSWV in perennial weeds. Following the completion of the harvesting season, farmers generally apply pesticides for agricultural husbandry. The roots of perennial weeds are exempted from agricultural cultivation and serve as a reservoir of viral inoculum for future cultivation. Each step can occur in either a preceding or succeeding manner. It replicates the cycle of disease spreading in a greenhouse.

Moreover, the presence of weeds in the greenhouse area makes it difficult to identify disease symptoms and requires further investigations (Wu et al., 2021), along with maintaining a disease-free environment before planting tomatoes in the upcoming season. Furthermore, the greenhouse surroundings are suitable for year-long cultivation considering the problem of limited time and the objective of meeting the demand for agricultural products within the country. However, agricultural husbandry is sometimes mismanaged. The key to novel herbicides for eliminating weed roots is nanotechnology, which has become a valuable resource for the agricultural industry as it has the potential to revolutionize current farming methods, including plant disease management and nanopesticides (Behl et al., 2022). A nanoherbicide is a tiny particle ranging from 1 to 100 nm in size that interacts with soil particles and eradicates weed seeds and weeds by targeting their roots (Manisankar et al., 2022). Currently, several researchers are attempting to develop nanoherbicides. SWFe-Imx/SWCS-Imx is a nanoherbicide that reduces herbicide release and demonstrates herbicidal activity comparable to that of commercial preparations (Cabrera et al., 2016). Our research indicates that managing weeds effectively is necessary to control and prevent viral infections in greenhouse tomato production. This discovery offers beneficial insights and a novel strategy for farmers and researchers.

Notes

Conflicts of Interest

No potential conflict of interest relevant to this article was reported.

Acknowledgments

This work was supported by the Korean Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET), Republic of Korea through Digital Breeding Technology Development Program (grant no. 322066-3). This work was supported by the Sungkyunkwan University and the BK21 FOUR (Graduate School Innovation) funded by the Ministry of Education (MOE, Korea).

Electronic Supplementary Material

Supplementary materials are available at The Plant Pathology Journal website (http://www.ppjonline.org/).

PPJ-OA-03-2024-0049-Supplementary-Fig-1.pdf

PPJ-OA-03-2024-0049-Supplementary-Fig-2.pdf

PPJ-OA-03-2024-0049-Supplementary-Fig-3,4.pdf

PPJ-OA-03-2024-0049-Supplementary-Fig-5,6.pdf

Fig. 1

Viral disease symptoms in tomatoes include: (A) tomato yellow leaf curl virus (TYLCV) with yellowing and curling leaves symptoms, (B) tomato spotted wilt virus (TSWV) with brownish ringspots and upward rolling of leaves symptoms, and (C, D) co-infection of TYLCV and TSWV symptoms.

Fig. 2

Weeds collected from tomato greenhouses for tomato yellow leaf curl virus or tomato spotted wilt virus detection: (A) Cerastium glomeratum, (B) Rorippa palustris, (C) Rumex crispus, (D) Senecio vulgaris, (E) Sonchus asper, and (F) Stellaria aquatica.

Fig. 3

Virus detection from the roots of six perennial weeds.

Fig. 4

Virus detection from the newly emerged leaves of Rumex crispus. The newly emerged leaves of R. crispus (n = 27) were collected and analyzed 28 days after the roots were transplanted on pots.

Fig. 5

Schematic diagrams of weed threats as continuous twindemic infections in tomato. The highlighted green color indicates healthy plants and nonviruliferous insects, including tomato, weed, whitefly, and thrips. The highlighted red color denotes infected plants and viruliferous insects, including tomato, weed, whitefly, and thrips. The highlighted black color refers to the reaction of pesticides and an area highlighted in brown indicates that it has been treated with pesticides. The light green, dark green, yellow, and gray colors represent the seasons of tomato planting, i.e., spring, summer, fall, and winter, respectively.

Table 1

The number of tomato plant and virus detection results in greenhouses

Plants	Symptoms	No. of samples	No. of detected		PDI (%)

			TYLCV	TSWV
Solanum lycopersicum	TSWV	15	0/50	15/50	30
	TYLCV	15	15/50	0/50	30
	TYLCV + TSWV	15	15/50	15/50	30
	No symptoms	5	1/50	0/50	-
Total no. of samples		50

TYLCV, tomato yellow leaf curl virus; TSWV, tomato spotted wilt virus; PDI, percentage disease incidence.

Table 2

List of weeds and virus detection results in tomato greenhouses

Plants	Growth cycle	No. of samples	No. of detected			PDI (%) of co-infection

			TYLCV	TSWV	TYLCV + TSWV
Bothriospermum zeylanicum	Annual	7	0/7	0/7	0/7	0
Cerastium glomeratum	Annual	16	6/16	2/16	2/16	12.5
Hemisteptia lyrata	Annual	3	0/3	0/3	0/3	0
Mazus pumilus	Annual	4	0/4	1/4	0/4	0
Oxalis corniculata	Perennial	12	0/12	0/12	0/12	0
Potentilla amurensis	Annual	3	0/3	0/3	0/3	0
Pseudognaphalium affine	Annual	4	0/4	0/4	0/4	0
Ranunculus sceleratus	Annual	4	0/4	0/4	0/4	0
Rorippa indica	Perennial	3	0/3	2/3	0/3	0
Rorippa palustris	Perennial	8	4/8	3/8	3/8	37.5
Rumex crispus	Perennial	38	10/38	12/38	10/38	26.31
Senecio vulgaris	Annual	6	6/6	3/6	3/6	50
Sonchus asper	Annual	6	4/6	2/6	2/6	33.33
Stellaria aquatica	Perennial	9	4/9	2/9	2/9	22.22
Trigonotis peduncularis	Annual	3	0/3	0/3	0/3	0
Veronica peregrina	Annual	4	0/4	0/4	0/4	0
Total no. of samples		210

TYLCV, tomato yellow leaf curl virus; TSWV, tomato spotted wilt virus; PDI, percentage disease incidence.

Table 3

Viral detection results from the roots of each weed

Plants	No. of samples	No. of detected			PDI (%) of co-infection

		TYLCV	TSWV	TYLCV + TSWV
Cerastium glomeratum	6	2/6	1/6	0/6	0
Rumex crispus	12	4/12	5/12	1/12	8.3
Rorippa palustris	6	2/6	1/6	1/6	16.66
Stellaria aquatica	12	4/12	8/12	3/12	25
Sonchus asper	6	5/6	4/6	3/6	50
Senecio vulgaris	6	3/6	3/6	2/6	33.33
Total no. of samples	48

TYLCV, tomato yellow leaf curl virus; TSWV, tomato spotted wilt virus; PDI, percentage disease incidence.

Table 4

Detection results of insect vectors Bemisia tabaci and Frankliniella occidentalis collected at the greenhouse

Insect vector	Scientific name	No. of samples	No. of detected

			TYLCV	TSWV
Whiteflies	Bemisia tabaci	8 (n = 40)	8/8	0/8
Thrips	Frankliniella occidentalis	8 (n = 40)	0/8	8/8
Total no. of samples		16 (n = 80)

TYLCV, tomato yellow leaf curl virus; TSWV, tomato spotted wilt virus.

Table 5

Statistical range analysis of TYLCV and TSWV detection results in newly apical leaves of Rumex crispus (n = 27 apical leaves)

	Range (n = 27)	Frequency of detection (n_i)	Mid points (m_i)	(n_i)(m_i)	Mean of detection
Group (TYLCV)
1	1-9	1	5	5	16.57^a
2	10-18	3	14	42
3	19-27	3	23	69
Group (TSWV)
1	1-9	0	0	0	23^a
2	10-18	0	0	0
3	19-27	1	23	23

TYLCV, tomato yellow leaf curl virus; TSWV, tomato spotted wilt virus.

^a Mean = Total of (n_i)(m_i)/total of frequency (n_i).

References

Abdelkhalek, A. and Hafez, E. 2020. Plant viral diseases in Egypt and their control. In: Cottage industry of biocontrol agents and their applications: practical aspects to deal biologically with pests and stresses facing strategic crops, eds. by N. El-Wakeil, M. Saleh and M. Abu-hashim, pp. 403-421. Springer International Publishing, New York, NY, USA.

Adkins, S. 2000. Tomato spotted wilt virus-positive steps towards negative success. Mol. Plant Pathol. 1:151-157.

Alda, L. M., Gogoaşă, I., Bordean, D.-M., Gergen, I., Alda, S., Moldovan, C. and Nita, L. 2009. Lycopene content of tomatoes and tomato products. J. Agroaliment. Processes Technol. 15:540-542.

Behl, T., Kaur, I., Sehgal, A., Singh, S., Sharma, N., Bhatia, S., Al-Harrasi, A. and Bungau, S. 2022. The dichotomy of nanotechnology as the cutting edge of agriculture: nano-farming as an asset versus nanotoxicity. Chemosphere 288:132533.

Bonassi, S., Coskun, E., Ceppi, M., Lando, C., Bolognesi, C., Burgaz, S., Holland, N., Kirsh-Volders, M., Knasmueller, S., Zeiger, E., Carnesoltas, D., Cavallo, D., da Silva, J., de Andrade, V. M., Demircigil, G. C., Odio, A. D., Donmez-Altuntas, H., Gattas, G., Giri, A., Giri, S., Gómez-Meda, B., Gómez-Arroyo, S., Hadjidekova, V., Haveric, A., Kamboj, M., Kurteshi, K., Martino-Roth, M. G., Montoya, R. M., Nersesyan, A., Pastor-Benito, S., Salvadori, D. MF., Shaposhnikova, A., Stopper, H., Thomas, P., Torres-Bugarín, O. ., Yadav, A. S., González, G. Z. and Fenech, M. 2011. The human micronucleus project on exfoliated buccal cells (HUMN(XL)): the role of life-style, host factors, occupational exposures, health status, and assay protocol. Mutat. Res. 728:88-97.

Bupi, N., Sangaraju, V. K., Phan, L. T., Lal, A., Vo, T. TB., Ho, P. T., Qureshi, M. A., Tabassum, M., Lee, S. and Manavalan, B. 2023. An effective integrated machine learning framework for identifying severity of Tomato yellow yeaf curl virus and their experimental validation. Research 6:0016.

Cabrera, A., Celis, R. and Hermosín, M. C. 2016. Imazamox-clay complexes with chitosan-and iron (iii)-modified smectites and their use in nanoformulations. Pest Manag. Sci. 72:1285-1294.

Chen, X., Cui, Z., Fan, M., Vitousek, P., Zhao, M., Ma, W., Wang, Z., Zhang, W., Yan, X., Yang, J., Deng, X., Gao, Q., Zhang, Q., Guo, S., Ren, J., Li, S., Ye, Y., Wang, Z., Huang, J., Tang, Q., Sun, Y., Peng, X., Zhang, J., He, M., Zhu, Y., Xue, J., Wang, G., Wu, L., An, N., Wu, L., Ma, L., Zhang, W. and Zhang, F. 2014. Producing more grain with lower environmental costs. Nature 514:486-489.

Costa, J. M. and Heuvelink, E. 2018. The global tomato industry. In: Tomatoes, eds. by E. Heuvelink, pp. 1-26. CABI, Wallingford, UK.

Czosnek, H. and Laterrot, H. 1997. A worldwide survey of Tomato yellow leaf curl viruses. Arch. Virol 142:1391-1406.

Davies, S. L. and Peoples, M. B. 2003. Identifying potential approaches to improve the reliability of terminating a lucerne pasture before cropping: a review. Aust. J. Exp. Agric. 43:429-447.

Dhaliwal, M. S., Jindal, S. K., Sharma, A. and Prasanna, H. C. 2020. Tomato yellow leaf curl virus disease of tomato and its management through resistance breeding: a review. J. Hortic. Sci. Biotechnol. 95:425-444.

Dorais, M., Ehret, D. L. and Papadopoulos, A. P. 2008. Tomato (Solanum lycopersicum) health components: from the seed to the consumer. Phytochem. Rev. 7:231-250.

Duffus, J. E. 1971. Role of weeds in the incidence of virus diseases. Annu. Rev. Phytopathol. 9:319-340.

Ferdinand, K. C., Nedunchezhian, S. and Reddy, T. K. 2020. The COVID-19 and influenza “twindemic”: barriers to influenza vaccination and potential acceptance of SARS-CoV2 vaccination in African Americans. J. Natl. Med. Assoc 112:681-687.

Ghanim, M., Morin, S., Zeidan, M. and Czosnek, H. 1998. Evidence for transovarial transmission of Tomato yellow leaf curl virus by its vector, the whitefly Bemisia tabaci. Virology 240:295-303.

Groves, R. L., Walgenbach, J. F., Moyer, J. W. and Kennedy, G. G. 2002. The role of weed hosts and tobacco thrips, Frankliniella fusca, in the epidemiology of Tomato spotted wilt virus. Plant Dis. 86:573-582.

Hanssen, IM., Lapidot, M. and Thomma, B. P. H. J. 2010. Emerging viral diseases of tomato crops. Mol. Plant-Microb. Interact. 23:539-548.

Hess, F. D. 2018. Herbicide absorption and translocation and their relationship to plant tolerances and susceptibility. In: Weed physiology, eds. by S. O. Duke, pp. 191-214. CRC Press, Boca Raton, FL, USA.

Hoang, N. H., Yang, H.-B. and Kang, B.-C. 2013. Identification and inheritance of a new source of resistance against Tomato spotted wilt virus (TSWV) in capsicum. Sci. Hortic 161:8-14.

Jones, D. R. 2003. Plant viruses transmitted by whiteflies. Eur. J. Plant Pathol. 109:195-219.

Karienye, J. M., Nduru, G. M., Huho, J. M. and Opiyo, F. E. 2018. Influence of rainfall variability on tomato production among small scale farmers in Kieni East Sub County. J. Arts Hum 8:7-19.

Kaushal, A., Sadashiva, A. T., Krishna Reddy, M., Srinivasa Rao, E., Singh, T. H., Sriram, S., Dhananjay, M. V., Venugopalan, R. and Ravishankar, K. V. 2020. Assessment of the effectiveness of Ty genes in tomato against Tomato leaf curl Bangalore virus. Plant Pathol. 69:1777-1786.

Kil, E.-J., Chung, Y.-J., Choi, H.-S., Lee, S. and Kim, C.-S. 2020. Life cycle-based host range analysis for Tomato spotted wilt virus in Korea. Plant Pathol. J. 36:67-75.

Kil, E.-J., Kim, S., Lee, Y.-J., Byun, H.-S., Park, J., Seo, H., Kim, C.-S., Shim, J.-K., Lee, J.-H., Kim, J.-K., Lee, K.-Y., Choi, H.-S. and Lee, S. 2016. Tomato yellow leaf curl virus (TYLCV-IL): a seed-transmissible geminivirus in tomatoes. Sci. Rep. 6:19013.

Kim, J.-H., Choi, G.-S., Kim, J.-S. and Choi, J.-K. 2004. Characterization of Tomato spotted wilt virus from paprika in Korea. Plant Pathol. J. 20:297-301.

Kokalis-Burelle, N. 2002. Biological control of tomato diseases. In: Biological control of crop diseases, eds. by S. S. Gnanamanickam, pp. 239-276. CRC Press, Boca Raton, FL, USA.

Kwon, Y., Cha, B. and Kim, M. 2022. Patterns of the occurrence of TYLCV and ToCV with whitefly on summer-cultivated tomato in greenhouse in Gwangju, Gyeonggi province. Res. Plant Dis. 28:39-45 (in Korean).

Lal, A., Pant, M. and Rani, A. 2015. The who’s who of plant viruses: a cognitive approach. Asian J. Pharm. Clin. Res. 8:60-68.

Lee, H., Song, W., Kwak, H.-R., Kim, J.-D., Park, J., Auh, C.-K., Kim, D.-H., Lee, K.-Y., Lee, S. and Choi, H.-S. 2010. Phylogenetic analysis and inflow route of Tomato yellow leaf curl virus (TYLCV) and Bemisia tabaci in Korea. Mol. Cells 30:467-476.

Lee, M., Uhm, K.-B., Lee, S., Park, H. and Ghanim, M. 2008. Occurrence of Bemisia tabaci and its vectored virus in Korea. In: Proceedings of the Korean Society of Applied Entomology Conference; pp 151-152 Korean Society of Applied Entomolohy, Suwon, Korea. (in Korean).

Li, J., Wang, J.-C., Ding, T.-B. and Chu, D. 2021. Synergistic effects of a Tomato chlorosis virus and Tomato yellow leaf curl virus mixed infection on host tomato plants and the whitefly vector. Front. Plant Sci. 12:672400.

Maes, P., Alkhovsky, S. V., Bào, Y., Beer, M., Birkhead, M., Briese, T., Buchmeier, M. J., Calisher, C. H., Charrel, R. N., Choi, IR., Clegg, C. S., de la Torre, J. C., Delwart, E., DeRisi, J. L., Di Bello, P. L., Di Serio, F., Digiaro, M., Dolja, V. V., Drosten, C., Druciarek, T. Z., Du, J., Ebihara, H., Elbeaino, T., Gergerich, R. C., Gillis, A. N., Gonzalez, J.-PJ., Haenni, A.-L., Hepojoki, J., Hetzel, U., Hồ, T., Hóng, N., Jain, R. K., van Vuren, P. J., Jin, Q., Jonson, M. G., Junglen, S., Keller, K. E., Kemp, A., Kipar, A., Kondov, N. O., Koonin, E. V., Kormelink, R., Korzyukov, Y., Krupovic, M., Lambert, A. J., Laney, A. G., LeBreton, M., Lukashevich, IS., Marklewitz, M., Markotter, W., Martelli, G. P., Martin, R. R., Mielke-Ehret, N., Mühlbach, H.-P., Navarro, B., Ng, T. F. F., Nunes, M. R. T., Palacios, G., Pawęska, J. T., Peters, C. J., Plyusnin, A., Radoshitzky, S. R., Romanowski, V., Salmenperä, P., Salvato, M. S., Sanfaçon, H., Sasaya, T., Schmaljohn, C., Schneider, B. S., Shirako, Y., Siddell, S., Sironen, T. A., Stenglein, M. D., Storm, N., Sudini, H., Tesh, R. B., Tzanetakis, IE., Uppala, M., Vapalahti, O. ., Vasilakis, N., Walker, P. J., Wáng, G., Wáng, L., Wáng, Y., Wèi, T., Wiley, M. R., Wolf, Y. I., Wolfe, N. D., Wú, Z., Xú, W., Yang, L., Yâng, Z., Yeh, S.-D., Zhâng, Y.-Z., Zhèng, Y., Zhou, X., Zhû, C., Zirkel, F. and Kuhn, J. H. 2018. Taxonomy of the family Arenaviridae and the order Bunyavirales: update 2018. Arch. Virol 163:2295-2310.

Mahy, B. W. J. and van Regenmortel, M. H. V. 2008. Encyclopedia of virology. 3rd ed. Elsevier Inc, Amsterdam, Netherlands. pp. 3192.

Manisankar, G., Ghosh, P., Malik, G. C. and Banerjee, M. 2022. Recent trends in chemical weed management: a review. Pharm. Innov. J. 11:745-753.

Massumi, H., Shaabanian, M., Pour, A. H., Heydarnejad, J. and Rahimian, H. 2009. Incidence of viruses infecting tomato and their natural hosts in the southeast and central regions of Iran. Plant Dis. 93:67-72.

Ong, S. N., Taheri, S., Othman, R. Y. and Teo, C. H. 2020. Viral disease of tomato crops (Solanum lycopersicum L.): an overview. J. Plant Dis. Prot. 127:725-739.

Palai, M. J. B. 2010. Weed management. Centurion University of Technology & Management, Paralakhemundi, Odisha, India. pp. 75.

Panno, S., Davino, S., Caruso, A. G., Bertacca, S., Crnogorac, A., Mandić, A., Noris, E. and Matić, S. 2021. A review of the most common and economically important diseases that undermine the cultivation of tomato crop in the mediterranean basin. Agronomy 11:2188.

Pék, Z., Helyes, L. and Lugasi, A. 2010. Color changes and antioxidant content of vine and postharvest-ripened tomato fruits. HortScience 45:466-468.

Pék, Z., Szuvandzsiev, P., Nemenyi, A., Helyes, L. and Lugasi, A. 2011. The effect of natural light on changes in antioxidant content and color parameters of vine-ripened tomato (Solanum lycopersicum L.) fruits. HortScience 46:583-585.

Polston, J. E., Schuster, D. J. and Taylor, J. E. 2009. Identification of weed reservoirs of tomato yellow leaf curl virus in Florida. In: 2009 Tomato Institute Proceedings by University of Florida, eds. by E. Simone, C. Snodgrass and M. Ozores-Hampton, pp. 32-33. University of Florida Institute of Food and Agricultural Sciences, Gainesville, FL, USA.

Rao, V. S. 2000. Principles of weed science. CRC Press, Boca Raton, FL, USA. pp. 564.

Riley, D. G., Joseph, S. V., Srinivasan, R. and Diffie, S. 2011. Thrips vectors of Tospoviruses. J. Integr. Pest Manag. 2:I1-I10.

Savary, S., Ficke, A., Aubertot, J.-N. and Hollier, C. 2012. Crop losses due to diseases and their implications for global food production losses and food security. Food Secur. 4:519-537.

Steinmann, H. H., Dickeduisberg, M. and Theuvsen, L. 2012. Uses and benefits of glyphosate in German arable farming. Crop Prot. 42:164-169.

Sundaraj, S., Srinivasan, R., Culbreath, A. K., Riley, D. G. and Pappu, H. R. 2014. Host plant resistance against tomato spotted wilt virus in peanut (Arachis hypogaea) and its impact on susceptibility to the virus, virus population genetics, and vector feeding behavior and survival. Phytopathology 104:202-210.

Wu, Z., Chen, Y., Zhao, B., Kang, X. and Ding, Y. 2021. Review of weed detection methods based on computer vision. Sensors 21:3647.

Zayan, S. A. 2019. Impact of climate change on plant diseases and IPM strategies. In: Plant diseases: current threats and management trends, eds. by S. Topolovec-Pintarić, IntechOpen, London, UK.