Turkish Journal of Agriculture and Forestry Turk J Agric For (2016) 40: 120-126 http://journals.tubitak.gov.tr/agriculture/ © TÜBİTAK Research Article doi:10.3906/tar-1410-113

New natural weed host raphanistrum L. () for Beet necrotic yellow vein virus and its vector Polymyxa betae Keskin

1 1 2 Nazlı Dide KUTLUK YILMAZ , Emine KAYA ALTOP , Colin James PHILLIPPO 1,* Hüsrev MENNAN 1 Department of Protection, Faculty of Agriculture, Ondokuz Mayıs University, Samsun, 2 Department of Horticulture, Michigan State University, East Lansing, MI, USA

Received: 28.10.2014 Accepted/Published Online: 06.08.2015 Final Version: 01.01.2016

Abstract: Rhizomania is an important virus disease of sugar beet (Beta vulgaris) caused by Beet necrotic yellow vein virus (BNYVV). The virus is transmitted to the roots of host by the plasmodiophorid Polymyxa betae. During survey studies in September and October 2009, yellow vein banding symptoms were observed on wild (Raphanus raphanistrum) plants growing in spinach (Spinacia oleracea) fields with a history of rhizomania in Samsun Province in the Black Sea Region of Turkey. To verify possible alternative hosts for BNYVV and P. betae, R. raphanistrum and spinach plants and soil samples were collected. BNYVV was detected in the samples of field-collectedR. raphanistrum using the DAS-ELISA test. This result was confirmed by RT-PCR and the Raphanus isolate was determined to be from the type A strain based on restriction fragment length polymorphism analysis. The presence of P. betae cystosori was not detected in the roots of R. raphanistrum plants using a light microscopy technique, but some objects resembling sporogenic plasmodia of P. betae were observed in the infested root cells. To confirm this result, total RNA was extracted from the roots of these samples and tested by RT-PCR using Polymyxa-specific primers. In contrast to the microscopy method, all samples tested positive for P. betae using RT-PCR. Healthy seeds of R. raphanistrum were planted in plastic pots containing soil from the same fields infested with BNYVV and the seedlings showed similar symptoms to those growing in natural conditions. To our knowledge this is the first report of a BNYVV infection of R. raphanistrum under natural conditions.

Key words: Rhizomania, Polymyxa betae, Brassicaceae, Raphanus raphanistrum, host

1. Introduction (Erdiller and Özgür, 1994; Kaya, 2009; Kutluk Yilmaz and Beet necrotic yellow vein virus (BNYVV), which is the Arli Sokmen, 2010). At present, the disease is controlled agent of rhizomania disease, is transmitted by Polymyxa by cultivating BNYVV-resistant sugar beet cultivars. betae Keskin and causes significant losses in sugar beet However, resistance-breaking BNYVV strains have been fields all over the world. The disease is characterized found in cultivars carrying the Rz1 gene in the United in sugar beet plants by extensive rootlet proliferation States, Europe (Liu and Lewellen, 2007; Bornemann et al., from the main taproot and leaf chlorosis. BNYVV is the 2015), and Turkey (Kutluk Yilmaz et al., 2012). type species of the genus Benyvirus and it contains four Both BNYVV and its vector P. betae have limited host or five ssRNAs (Koenig et al., 1986). Molecular analyses ranges. A few members belonging to Amaranthaceae, (restriction fragment length polymorphism, single- Asteraceae, Caryophyllaceae, Chenopodiaceae, strand conformation polymorphism, and sequencing) of Portulacaceae, Solanaceae, and Poaceae were recorded as BNYVV isolates have revealed the existence of three strain alternative hosts for BNYVV (Tamada and Baba, 1973; Barr groups (A, B, and P type) in Europe (Kruse et al., 1994; and Asher, 1992; Hugo et al., 1996; Legreve et. al., 2005; Koenig et al., 1995; Koenig and Lennefors, 2000; Ward et Mouhanna et al., 2008). Until now, BNYVV host studies on al., 2007). Rhizomania disease was first reported in sugar Brassicaceae, such as Raphanus raphanistrum L., Raphanus beet in Turkey in 1987 (Koch, 1987). Since then it has sativus L., napus L., and arvensis L., have spread to many major sugar beet-growing areas within failed to demonstrate infection by this virus or its vector the country, especially the Western and Central Black Sea (Barr and Asher, 1992; Hugo et al., 1996; Legreve et al., 2005). Region and the Central Anatolia and Marmara Regions R. raphanistrum (wild radish) is a winter annual species

* Correspondence: [email protected] 120 KUTLUK YILMAZ et al. / Turk J Agric For belonging to Brassicaceae. The broad ecological tolerance Plant Protection Department collection) were used of these weeds, such as quick germination, rapid growth, to grow sugar beets in a bait plant test as positive and and abundant seed production, make it a successful weed in negative controls in serological and molecular studies. many countries. This weed, a native of northern Europe and Samples were homogenized and used for bioassays under northern Asia, is found in cultivated fields around the world greenhouse conditions. (Cheam and Code, 1995; Blackshaw, 2001). 2.2. Seed material A major problem is that the virus particles survive Seeds from Chenopodiaceae, Brassicaceae, inside resting spores of P. betae for at least 15 years (Abe Amaranthaceae, and Portulacaceae were used in this and Tamada, 1986). In this long period, some weed species study and supplied from Ondokuz Mayıs University, Plant act as alternative hosts for the virus and vector (Hugo Protection Department collection. et al., 1996; Legreve et al., 2005; Mouhanna et al., 2008; 2.3. Bait plant technique Rysanek et al., 2008). In 2009, several spinach fields with a Seeds were sown separately into 500-mL plastic pots history of rhizomania in Samsun Province in the Black Sea containing rhizomania-infested soil in three replications Region of Turkey exhibited yellow vein banding symptoms with ten seeds per plastic pot. The pots were placed in a in wild radish and yellow-green mottling in spinach . controlled glasshouse at 19 °C (night) and 25 °C (day). The presence of these kinds of virus symptoms on plants After 8 weeks, plants were harvested and their roots were in these fields raises the question of where BNYVV is carefully washed in running tap water. One section of the present in wild radish and spinach crops. Like sugar beet, roots from each bait plant was checked for the presence of spinach (Spinacia oleracea L.) also belongs to the family P. betae cystosori by light microscope. Another part was Chenopodiaceae. In , BNYVV was identified in tested for the presence of BNYVV by ELISA. Plants that spinach plants in 1995 (Autonel et al., 1995) and then the tested positive for BNYVV or P. betae were assessed again virus was found in spinach crops in in 2010 (Liu in a second test to determine if the virus and vector could et al., 2010). More recently, BNYVV was also detected in be transmitted back to sugar beets. For this purpose, dried field-grown spinach in the western part of Turkey (Gumus roots of infected plants were cut into small fragments and et al., 2014). mixed with equal parts sterile sand and soil, into which Improved control strategies for BNYVV require further a BNYVV-susceptible sugar beet seed (Arosa) was sown. knowledge of potential reservoirs of infection in sugar After 8 weeks, infections by P. betae and BNYVV were beet fields. However, reports on natural hosts of BNYVV assessed. based on large-scale surveys are scarce. Maintenance of Besides this, sugar beet seeds of rhizomania-susceptible virus inoculum in weed species might provide a means Arosa were planted into 300-mL pots of three BNYVV- for viruses to survive through the seasonal cycle and and P. betae-infested soils. Noninfested soil samples mixed explain their prevalence during epidemics. Therefore, with sterile sand (1:1) were used as positive and negative discovering the weed reservoirs of BNYVV may help controls in serological and molecular studies. us understand their epidemiology and provide basic information for implementation of effective management 2.4. Serological tests strategies. Therefore, the objective of this study was to test DAS-ELISA was performed for BNYVV using polyclonal R. raphanistrum for reservoirs of BNYVV and its vector P. antiserum (Sediag, ) according to Clark and Adams betae by using ELISA and RT-PCR tests. (1977). A sample was considered positive when the absorbance value at 405 nm was more than two times the 2. Materials and methods mean of the negative controls (Meunier et al., 2003). 2.1. Plant and soil material 2.5. Microscopy analysis of Polymyxa betae In 2009, typical virus-like symptoms such as yellow-green Root samples were stained with lactophenol containing mottling in spinach plants and yellow vein banding on 0.1% acid fuchsin and examined under a light microscope R. raphanistrum were observed in a spinach field in the (Leica, ) to detect the presence of P. betae Bafra district of Samsun Province in Turkey. This area cystosori (Abe and Tamada, 1986). is well known from our previous studies to be highly 2.6. Total RNA isolation contaminated by P. betae and BNYVV. For this reason, Total RNA was extracted from the roots and leaves of a total of 25 R. raphanistrum and 9 spinach plants were R. raphanistrum and the roots of sugar beet that had collected. Soil samples were also taken from patches with previously tested positive for BNYVV in ELISA. Total plants displaying typical virus symptoms. Additionally, RNA was extracted from 100 mg of frozen tissue using three soils infested with P. betae and BNYVV and a the RNeasy Mini Kit (QIAGEN, USA) according to the noninfested soil (from the Ondokuz Mayıs University, manufacturer’s instructions.

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2.7. RT-PCR for detection of BNYVV and Polymyxa 2.8. Restriction fragment length polymorphism analysis betae To produce a PCR product for restriction fragment length One-step RT-PCR was performed as described in the polymorphism (RFLP) analysis, the primers proposed manufacturer’s manual (QIAGEN). For the detection of by Kruse et al. (1994) were used to amplify the RNA-3 BNYVV, the primers proposed by Kruse et al. (1994) were as described above, which was then cut separately with used (forward primer: GTGATATATATGTGAGGACGCT enzymes EcoRI, BamHI, and MspI. and reverse primer: CCGTGAAATCACGTGTAGTT), which amplified RNA-3. The RT-PCR reaction included 3. Results and discussion the following: 15.5 µL of RNase-free water, 1 µL of dNTPs 3.1. Detection of BNYVV mix (400 µM), 5 µL of 5X Q solution, 0.25 µL each of A total of 9 spinach plants and 25 R. raphanistrum samples forward and reverse primers (0.6 µM), 1 µL of QIAGEN were collected from the spinach field and tested by DAS- OneStep RT-PCR enzyme mix, and 2 µL of RNA sample. ELISA. In this study, BNYVV was detected in 20 samples Reverse transcription consisted of 30 min at 50 °C and of R. raphanistrum collected from the field (Figure 1A). 15 min at 95 °C. We then ran 35 cycles composed of: On the other hand, 7 of the field-grown spinach samples denaturation for 30 s at 94 °C, annealing for 30 s at 53 °C, were infected with BNYVV. Generally, BNYVV-positive and elongation for 1 min at 72 °C. A final elongation of 7 spinach plants have yellow-green mottling symptom on min at 72 °C was also performed. their leaves. Although spinach is grown in many regions For the detection of Polymyxa in roots of R. raphanistrum of Turkey, BNYVV has only been recently recorded in and sugar beet, oligonucleotides flanking 5.8s-rDNA and spinach production areas in İzmir and Manisa provinces internal transcribed spacers (ITS) were used. This includes located in the western part of Turkey (Gumus et al., 2014). In the ELISA tests with the R. raphanistrum samples, upstream 5`-GAGGCATGCTTCCGAGGGCTCT-3` color development was very slow. For this reason, the and downstream primer 5`-CTGCGGAAGGAT- DAS-ELISA test microtiter plates were read following CATTAGCGTT-3`(Ward and Adams, 1998). The RT-PCR 2 h substrate incubation at room temperature and then reaction included the following: 15.2 µL of RNase-free again following overnight substrate incubation at 4 °C. water, 1 µL of dNTPs mix (400 µM), 5 µL of 5X Q solution, Even after overnight incubations, relatively low A405 0.15 µL each of forward and reverse primers (0.6 µM), 1 values were obtained for most of the samples. The positive µL of QIAGEN OneStep RT-PCR Enzyme mix, and 2.5 controls supplied by the Sediag Biochemica and root µL of RNA sample. Reaction conditions were the same as samples from the sugar beet bait plants grown in BNYVV- used for the amplification of RNA-3 except for annealing infested soils gave satisfactory positive results at 2 h and temperature (55 °C). 16 h substrate incubations. The reason for the low ELISA After PCR amplification, 2 µL of loading buffer was values could be low virus titers in R. raphanistrum samples. added to the PCR products. The samples were analyzed In addition, RT-PCR assay to detect BNYVV and P. betae on 1.2% ethidium bromide agarose gel in 1X Tris-borate- was carried out on total RNA extracted from leaves and EDTA buffer using the Gel Doc 2000 Systems (Bio-Rad, root samples from R. raphanistrum and root samples from USA) (Ward and Adams, 1998). the sugar beet bait. All samples tested positive for BNYVV

A B Figure 1. Yellow vein banding in field-grown (A) and greenhouse-grown (B) BNYVV- infected Raphanus raphanistrum.

122 KUTLUK YILMAZ et al. / Turk J Agric For in this RT-PCR detection assay (Figure 2). Until now, M 1 2 3 4 5 6 7 8 BNYVV host studies on Brassicaceae members such as R. raphanistrum, R. sativus, B. napus, and S. arvensis have failed to demonstrate infection by this virus or its vector (Barr and Asher, 1992; Hugo et al., 1996; Legreve et al., 2005). However, BNYVV and P. betae were both detected in Capsella bursa-pastoris (L.) Medik. and Thlaspi arvense L., which belong to the family Brassicaceae, in a bait plant test using RT-PCR by Legreve et al. (2005). However, in this study, neither BNYVV nor its vector were found in these species by ELISA or light microscopy, respectively (Legreve et al., 2005). Later on, Mouhanna et al. (2008) found that Figure 3. The results of RFLP analysis of the RT-PCR products C. bursa-pastoris was infested with P. betae and BNYVV of BNYVV RNA-3. M: 1-kb ladder (Promega, USA). The arrow using bait plant test and back transmission studies. To our indicates the BNYVV RNA-3-specific 1218-bp DNA fragment. knowledge, this is the first report of a BNYVV infection of Lanes 1–4: RNA from roots of R. raphanistrum (EcoR1, BamH1, R. raphanistrum in the family Brassicaceae under natural Msp1, and uncut PCR product). Lanes 5–8: RNA from the conditions. roots of the bait sugar beet plant grown in BNYVV-infested soil (EcoR1, BamH1, Msp1, and uncut PCR product). 3.2. RFLP analysis of BNYVV Kruse et al. (1994) showed that RNA-3 of B type strains is cut by BamHI, EcoRI, StyI, and MspI. In contrast, the objects resembling sporogenic plasmodia of P. betae were A type strains were not cut by any of these restriction described in the root cells of R. raphanistrum (Figure 4). To enzymes. Our BNYVV isolates were not digested by these confirm this result, sugar beet bait plant and field-grown restriction enzymes, suggesting that the isolate from R. R. raphanistrum samples were subjected to RT-PCR assay raphanistrum belongs to the A type strain (Figure 3). with Polymyxa-specific primers (Ward and Adams, 1998) The A type, the most widespread strain (Schirmer et al., to determine whether they were infected with Polymyxa 2005), has been previously detected in Turkey (Kruse et al., spp. Ward and Adams (1998) emphasized that the 1994; Kutluk Yilmaz et al., 2007); the B type strain is more products of amplification using these primers on isolates restricted and mainly found in , France (Kruse of Polymyxa graminis types I and II and P. betae could all et al., 1994; Koenig et al., 2008), Japan (Miyanishi et al., be distinguished from one another on 1.2% agarose gels. 1999), and (Li et al., 2008). Therefore, a 250-bp DNA fragment expected for the ITS 3.3. Detection of P. betae in infested roots Roots of R. raphanistrum that were collected from a spinach field were inspected for the presence of P. betae using light microscopy. P. betae cystosori were not detected in roots of any of these plants, though some

M 1 2 3 4

1500-bp

1000-bp 750-bp

Figure 2. Confirmation of the presence of BNYVV in leaves of R. raphanistrum using RT-PCR analysis. M: DNA size marker. The arrow indicates the BNYVV-specific 1218-bp DNA fragment. Lane 1: negative control (water). Lane 2: BNYVV-containing R. raphanistrum leaves. Lane 3: healthy control (noninfected sugar beet cultivar Arosa). Lane 4: positive control (sugar beet infected Figure 4. P. betae sporogenic plasmodia in root cells of R. with BNYVV). raphanistrum.

123 KUTLUK YILMAZ et al. / Turk J Agric For between the 5.8 s and the 18 s rRNA of P. betae was obtained factors, or insufficient nutrients, which might have limited from the roots of the susceptible sugar beet cultivar grown the development of pathogen structures (Barr et al., 1995). in rhizomania-infested soils and R. raphanistrum in this 3.4. Bait plant and back inoculation tests assay (Figure 5). Rysanek et al. (2008) suggested that the Among all Brassicaceae species tested for P. betae and PCR method would be useful for verifying the presence BNYVV host status, sugar beet, spinach, Chenopodium of P. betae in the case of low numbers of cystosori in host album L., Chenopodium murale L., Amaranthus retroflexus range studies. Moreover, in some plant species, plasmodia L., and Portulaca oleracea L. were added to the bait plant or zoosporangia could be present without cystosori; PCR test. The Table shows the species that became infected with could reveal such hidden infections. Similarly, Legreve et either P. betae or BNYVV. In this study, P. betae cystosori al. (2008) found that BNYVV and P. betae were detected were found by light microscopy only in sugar beet, C. by RT-PCR in some species belonging to different album, A. retroflexus, and P. oleracea. Interestingly, we families, while the virus and vector went undetected by could not detect either P. betae or BNYVV in spinach ELISA and microscopy, respectively. We suggest that plants. From all tested species, infection by BNYVV was viruliferous P. betae zoospores were able to penetrate the only detected in sugar beet and R. raphanistrum using roots of R. raphanistrum plants and introduce the virus, ELISA. R. raphanistrum plants also showed symptoms of but the protozoa were unable to produce cystosori and yellow vein banding under controlled conditions similar complete their life cycle in the roots of wild radish. An to natural conditions (Figure 1B). However, P. betae was incomplete infection, similar to our result, was reported not detected using the light microscopy technique in these with P. betae in Gomphrena globosa (Al Musa and Mink, plant roots. Neither BNYVV nor P. betae was detected in 1981) and P. graminis in Arachis hypogaea (groundnut) the other tested Brassicaceae species: R. sativus, E. sativa, (Thouvenel and Fauquet, 1981). Later on, Barr et al. B. napus, B. rapa, B. juncea, and B. oleracea (Table). (1995) reported similar observations about early P. betae Besides this, we tried using roots of a plant infected developmental stages without cystosori in two wild beet with a vector or the virus (sugar beet, C. album, A. species, Beta patellaris and Beta procumbens. Although the retroflexus, P. oleracea, and R. raphanistrum) to see if they wild radish leaves became infected naturally with BNYVV had the potential to act as an alternative host for P. betae in the field, they were unable to provide sources of virus and BNYVV to transmit to susceptible sugar beet plants. infection to other plants, whereas comparable sugar beet Transmission back to sugar beet occurred only from B. roots were able to do so. There are various factors, such vulgaris (Table). as environmental conditions, the lack of vital nutritional In rhizomania disease, the virus–vector–host relationships are very complex. Over the last decades, the M 1 2 3 4 5 existence of different genotypic groups has become obvious, and they correspond to differences in pathogenicity and specific geographical region. BNYVV isolates fall into three strain groups: A, B, and P types (Kruse et al., 1994; Koenig et al., 1995; Koenig and Lennefors, 2000; Ward et al., 2007). In the last years, resistance-breaking BNYVV strains have been found in cultivars carrying the Rz1 gene in the United States, Europe (Liu and Lewellen, 2007; 1000- bp Bornemann et al., 2015), and Turkey (Kutluk Yilmaz et al., 750- bp 2013). Beside this, the performance of viruliferous P. betae in soils is influenced by many different biotic and abiotic 500- bp factors. Gerik and Duffus (1988) described differences in vectoring abilities of P. betae populations depending on their origin. There are also subgroups of P. betae with specific host ranges, such as betae, amaranthi, and the family Portulacaceae (Barr, 1979; Abe and Tamada, 1986; Abe and Ui, 1986). Moreover, weeds are populations with a wide range of genetic variability. In this respect, this appears to be the first report of R. raphanistrum Figure 5. RT-PCR analysis for detection of P. betae. M: 1-kb DNA ladder (Promega). The arrow indicates the P. betae-specific 250- from the family Brassicaceae acting as a host of BNYVV bp DNA fragment. Lane 1: root RNA from R. raphanistrum. Lane under natural conditions. The Raphanus isolate was 2: negative control (healthy sugar beet roots). Lanes 3–5: positive assigned to the A type strain based on RFLP analysis. controls (BNYVV-containing sugar beet roots). Although P. betae cystosori were not found in the roots of

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Table. ELISA and microscopy results for the bait plant and back inoculation to B. vulgaris tests.

Presence of P. betae Infection by Back inoculation Family Plant species cystosori (microscopy) BNYVV (ELISA) B. vulgaris Chenopodiaceae Beta vulgaris L. cv. Arosa + + + Chenopodiaceae Spinacia oleracea L. - - nt Chenopodiaceae Chenopodium album L. + - - Chenopodiaceae Chenopodium murale L. - - nt Amaranthaceae Amaranthus retroflexus L. + - - Portulacaceae Portulaca oleracea L. + - - Brassicaceae Raphanus raphanistrum L. - + - Brassicaceae Raphanus sativus L. - - nt Brassicaceae Brassica napus L. (B-12 accession) - - nt Brassicaceae Brassica napus (B-2 accession) - - nt Brassicaceae Brassica juncea (L.) Czern (B-4 accession) - - nt Brassicaceae Brassica juncea (B-7 accession) - - nt Brassicaceae Brassica juncea (B-5 accession) - - nt Brassicaceae Brassica oleracea L. - - nt Brassicaceae Brassica rapa L. - - nt Brassicaceae Eruca sativa L. - - nt nt: not tested.

R. raphanistrum, it may infect wild radish roots without host of BNYVV may help to understand its epidemiology cystosori development. Therefore, R. raphanistrum seems and could provide basic information for implementation to be a relatively inefficient host for rhizomania compared of effective management strategies. Further work is needed to sugar beet. Nonetheless, knowing that this weed is a in the search for natural hosts of BNYVV and P. betae.

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