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Pak. J. Nematol.

, 30 (2): 115-127, 2012

OCCURRENCE AND INTERACTIONS AMONG SUGAR BEET CYST NEMATODE AND BEET SOILBORNE VIRUSES IN NORTHERN TURKEY
S. MENNAN*, N.D.K. YILMAZ*, G. AYDINLI** AND S. ANKAYA* Ondokuz Mayis University, Agricultural Faculty, Plant Protection Department, 55139 Atakum-Samsun, Turkey
*Corresponding authors email: smennan@omu.edu.tr
Abstract A survey was conducted to determine the distribution of Beet necrotic yellow vein virus (BNYVV), Beet soilborne virus (BSBV), their vector Polymyxa betae and sugar beet cyst nematode (Heterodera schachtii Schm.) (SBN) in sugar beet fields in northern parts of Turkey. A total of 200 soil samples were collected at random from fields in Samsun, Amasya, Tokat, Corum, and Cankr provinces during 2004 and 2005 growing seasons. The results of ELISA tests showed that BSBV was the most prevailing virus (40.5%), followed by BNYVV (27.5%) in the regions. Of the 200 fields surveyed, 92 samples infested by at least one virus (46%), 55 samples infested by SBN (27.5%) and 161 samples infested by viruliferous or aviruliferous P. betae (80.5%). In the mixed infections, the combination BNYVV and BSBV was the most frequent (15%) followed by aviruliferous P. betae+SBN (8.5%). Also, the number of nematode cysts was significantly lower in BNYVV+P. betae, BSBV+P. betae, BNYVV+BSBV+P. betae and aviruliferous P. betae compared with healthy samples.
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Sugar beet (Beta vulgaris L.) is mainly affected by soilborne viruses such as Beet necrotic yellow vein virus (BNYVV) and Beet soilborne virus (BSBV), in addition to sugar beet cyst nematode (Heterodera schachtii Schm.) (SBN) in many countries (Meunier et al., 2003). Both nematode and viruses generally cause root proliferation and chlorosis symptoms on the susceptible sugar beet cultivars as well as severe yield losses and decreases sugar content (Griffin, 1981; Whitney & Duffus, 1986). Indeed, the loss in sugar beet production attributed to nematodes is estimated to be 10% and only SBN accounts for more than 90% of that loss (Whitney & Duffus, 1986) and estimated average root yield loss caused by the SBN was 6.95 t ha-1 (Cook, 2008). Because of great variation in climatic and environmental conditions under which sugar beets are grown, there is considerable variation in the degree of pathogenicity of SBN on sugar beet (Griffin, 1981). As coming to viruses, only BNYVV causes almost 100% yield losses. While these viruses infect members of Chenopodiaceae family, SBN has a rather wide host range in addition to sugar beet (Whitney & Duffus, 1986). These viruses are transmitted by plasmodiophorid Polymyxa betae
** Bozok University, Agricultural Faculty, Plant Protection Department, 66200 Yozgat, Turkey.

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Keskin, ensuring their persistence in the soil over a long period (Asher, 1999; Prillwitz & Schlsser, 1992). Moreover, their vector is distributed even more widely than the viruses (Brunt & Richards, 1989). The occurrence of two different viruses transmitted by a similar vector and SBN, increases many questions regarding to their interactions in the same sugar beet plant. Our knowledge is not enough to explain such kind of interaction due to limited study (Mouhanna et al., 2001). However, this study is giving more opportunities to learn about how such kind of interaction occur under green house conditions. Despite this information, there is no data about the naturally infested soil with these all agents. Therefore, the objectives of this study were to determine (i) the incidences of BNYVV, BSBV, P. betae and SBN in sugar beet fields, and (ii) the effects P. betae, BNYVV, BSBV and SBN either alone or in combination on mean ELISA values on sugar beet in naturally infested soils from northern parts of Turkey. Materials and Methods Soil sampling: A total of 200 soil samples were collected at random from sugar beet fields in Samsun, Amasya, Tokat, Corum, and Cankr provinces of northern parts of Turkey during 2004 and 2005 growing seasons (Fig. 1). In each field, composite sampling was obtained by randomly taking five to twelve subsamples at the depth of 20 cm of soil.

Fig. 1. Surveyed areas from sugar beet fields of northern parts of Turkey.

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Extraction of cysts from soil : Cysts were extracted from air-dried soil samples by a sieving method, using the remainder of each composite sample that had been placed in an open paper bag and allowed to air dry thoroughly for about 2 weeks, and then a 1000 g subsample of dry soil was used to extract the cysts. A sieving method was used to obtain the cysts, which were counted in 6 cm diameter plastic petri dishes after collection with a coated sable skin brush under a stereo binocular microscope (Leica, S6D). Juveniles for morphological observation were recovered from cysts incubated in water in watch glasses. Juveniles were fixed in 3% formaldehyde solution and processed to glycerin by the formalinglycerin method (Hooper, 1986; Golden, 1990). Cysts were fixed in 3% formaldehyde solution. The vulval cone region was excised and embedded in a heated drop of glycerin jelly on a cover slip and sealed between cover slips held in aluminum holders. The morphological and morphometric characters examined with a light microscope and nematodes identified through use of taxonomic keys (Golden, 1986). Later, the cysts of SBN were counted in soil samples. Bait plant technique: The soil samples collected during surveys were used in bait plant tests. All soil samples were dried at room temperature. After all stones and visible roots were removed from the soil samples, each sample was ground using a wooden hammer and collected through a 2 mm aperture sieve for bait plant tests. Each of the soil samples were diluted to 1:1 vol/vol with autoclaved sand. Then, 10 sugar beet seeds of the rhizomania-susceptible cultivar (cv. Arosa) were sown in 300 ml plastic pots containing mixed soil. Each soil sample was used in duplicate. The plants were grown under controlled conditions with a 12-h photoperiod at 20C (night) and 23C (day). All plants were watered with Hoaglands solution directly as needed. After harvest, whole root systems were carefully collected in running tap water, the number of plants per pot were determined and all plants within a pot were combined and their roots were used (i) to determine the presence of BNYVV and BSBV by ELISA (ii) occurrence of P. betae cystosori and, (iii) to detect SBN infection by staining with acid fuchsine. Plant material was stored at -20C until used for testing. Serological tests: The roots of sugar beet plants were tested for presence of BNYVV and BSBV by ELISA. The double-antibody sandwich (DAS)-ELISA was used to determine BNYVV infection by using antiserum supplied by Sediag Biochemica (France). DAS-ELISA was performed according to Clark & Adams (1977), except that extraction buffer included 0.1 % non-fat dry milk instead of bovine serum albumin (Arif et al., 1994). A triple- antibody sandwich (TAS)ELISA was used to test for BSBV. TAS-ELISA was performed according to instructions of the antiserum producer (Adgen, UK). The plates were measured using a microplate reader (Tecan Spectra II, Grdig/Salzburg, Austria). All reported ELISA values were of 2 h period and samples were considered positive

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when the absorbance at A405 nm values exceeded the mean of the healthy controls by least factor of two (Meunier et al., 2003). Detection of SBN infection and P. betae: Root samples taken from bait plants were stained with 0.1% acid fuchsine. Each root system checked for infection and development of SBN under a stereobinocular microscope (Olymphus SZ61) and a light microscope (Olymphus CX41) (Hussey, 1985). In addition to that, the resting spore clusters of P. betae were investigated in their roots under a light microscope (Leica, Switzerland) (Abe & Tamada, 1986). Statistical analysis: Kendalls tau rank correlation analysis was used to determinate the relationship between BNYVV, BSBV, P. betae and SBN either alone or in combination, on sugar beet. Also, Pearson correlation analysis was performed to determinate the relationship between mean ELISA values of BNYVV and BSBV on sugar beet in naturally infested soil. All the computational work was performed by means of MINITAB (Minitab V. 13.20, 2000). Results and Discussion Detection of SBN and soilborne viruses in sugar beet fields: A total of 200 fields were surveyed in Samsun, Amasya, Tokat, Corum and Cankiri provinces in Turkey. The results of serological tests, soil extraction and also stained studies showed that sugar beet fields in northern parts of Turkey were infested with BNYVV, BSBV, P. betae and SBN. The incidences of these viruses, their vector and also SBN, alone and in combination, in the soil samples are shown in Table 1a, b. Of the 200 fields surveyed, 55 samples infested by SBN (27.5%), 92 samples infested by at least one virus (46%), and 161 samples infested by viruliferous or aviruliferous P. betae (80.5%). Single infection rates were found 3.5%, 28%, 5.5% and 26% for BNYVV, BSBV, SBN and aviruliferous P. betae, respectively. According to ELISA results, BSBV was the most prevailing virus (40.5%), followed by BNYVV (27.5%) in the regions. Moreover, BSBV was the predominant virus in all provinces surveyed except Corum province. Mixed infections were detected in 34.5% of samples. In the mixed infections, the combination BNYVV and BSBV was the most frequent (15%) followed by aviruliferous P. betae + SBN (8.5%), BNYVV + BSBV + SBN (7%), BSBV + SBN (4.5%) and BNYVV + SBN (2%) (Table 1b). Besides this, according to the results of soil extractions, fields were detected with SBN densities ranging 2-34 cysts per 100 g of soil. Six weeks after planting at bait plant test, different life stages of SBN (Fig. 2A-C) and also P. betae cystosori (Fig. 2D) were observed in the root samples. Thus, it was confirmed that this period was enough to see a few to many adult females attached to fibrous sugar beet roots like Steele (1995).

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Table 1a. The presences of BNYVV, BSBV, P. betae and SBN in sugarbeet fields in northern parts of Turkey.
Province/ District No. of fields Healthy Number of fields that has single infections Viruliferous P. betae Aviruliferous SBN P. betae BNYVV BSBV

Centrum Tasova Gumushacikoy Suluova Merzifon Gyncek Merkez Total Pazar Niksar Erbaa Turhal Zile Total Carsamba Havza Bafra Vezirkopru Ladik Kavak Total Osmanck Sungurlu skilip Alaca Lain Total Kzlrmak Total Grand total

9 14 11 14 12 11 15 86 10 13 11 9 10 53 15 6 18 4 4 1 48 3 2 1 1 1 8 5 5 200

1 0 1 2 4 1 2 11 (12.8) 3 3 0 3 2 11 (20.8) 3 1 2 0 0 0 6 (12.5) 0 0 0 0 0 0 (0) 0 0 (0) 28 (14%)

Amasya 0 2 0 2 0 5 1 5 0 2 0 1 0 0 1 (1.2) 17 (19.8) Tokat 1 1 2 0 1 1 0 1 0 2 4 (7.6) 5 (9.4) Samsun 0 1 0 2 0 1 0 0 0 0 0 0 0 (0.0) 4 (8.3) orum 1 0 0 0 0 0 0 0 0 0 1 (12,5) 0 (0) ankr 1 2 1 (20) 2 (40) 7 (3.5%) 28 (14%)

1 1 1 2 3 0 3 11 (11.8) 2 3 0 2 1 8 (15.1) 10 1 13 3 2 0 29 (60.4) 1 2 0 1 0 4 (50) 0 0 (0) 52 (26%)

1 0 2 1 0 0 1 5 (5.8) 1 0 0 1 1 3 (5.7) 0 1 1 0 0 0 2 (4.2) 0 0 0 0 0 0 (0) 1 1 (20) 11 (5.5%)

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The current study was the first to show the provincial distribution of SBN in northern parts of Turkey. Presence of the SBN in Turkey was the first reported in Babaeski district in Trakya region by Diker in sugar beet growing areas in 1959. Later on, in 1969, it was recorded in all of Trakya region and then it was found in Eskisehir province in Turkey (Susurluk & kten, 1999). BNYVV has been reported frequently in Turkey since 1990s (Vardar & Erkan, 1992). Although the presence of BNYVV and BSBV were known in some of these regions (Erdiller & zgr, 1994; Meunier et al., 2003; Kutluk-Yilmaz et al., 2004; 2005; zer & Ertun, 2005; Kutluk Yilmaz et al., 2007). The results of our surveys revealed that sugar beet fields were infected with an average rate of 46% (92 of the samples) with soilborne viruses transmitted by P. betae and 27.5% (55 of the samples) with SBN. Also, 27 samples (3.5%) infested with SBN and soilborne viruses together. Unfortunately, growers in the region are not aware of how soilborne viruses and SBN spread from field to field and do not know about precautions to control viruses and SBN transmission. The widespread occurrence of soilborne viruses and SBN in the region is possibly due to the use of highly contaminated agricultural tools when preparing their field. Also, irrigation water, use of sewage and waste resulting from sugar beet factories could be important dissemination factors of the diseases and SBN to new areas (Tuitert & Hofmeester, 1992). Interactions among BNYVV, BSBV, P. betae, SBN and symptoms expression on bait plants: Additionally, bait plant studies were led to clarify to us the effects of P. betae, BNYVV, BSBV and SBN, alone and in combination, on mean ELISA values in sugar beet in naturally infested soil (Table 2). ELISA values obtained from BNYVV were significantly increased when BNYVV+BSBV+P. betae, BNYVV+ P. betae+SBN and BNYVV+BSBV+ P. betae+SBN were found as mixed infections (P<0.01) but not alone with BSBV+ P. betae. Besides this, absorbance values of BSBV were also significantly increased by BNYVV+BSBV+P. betae, BSBV+ P. betae+SBN and BNYVV+ BSBV+ P. betae+ SBN were found as mixed infections (P<0.01). Also, there was negative correlation between aviruliferous+viruliferous P. betae (P<0.01) and BSBV ELISA absorbance values (P<0.05) (Table 2). In some cases, plant parasitic nematodes may negatively affect under biotic and abiotic factors including some fungi and bacteria in nature as a result of this inhibition, these kind of soils are known as suppressive soils. Sugar beet fields commonly have a complex microbial community including viruses, their vector P. betae and also nematodes. As a result of that, inconsistent interactions could be appearing. For fungal diseases, there have been synergism between SBN and some soil fungi for example Rhizoctonia solani (Polchronopoulos el al., 1969),

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Pythium ultimum (Whitney, 1974), Janowiez et al., (1994) showed this negative interaction between Rhizoctonia solani Kuhn and Globodera rostochiensis Woll in potatoes fields. As coming to viruses, Beet mosaic virus (BMV) has important effect on SBN, but beet yellowing viruses reduced the number of cysts per plant by 75% (Schlsser, 1962). In our study, the number of nematode cysts was significantly lower when BNYVV+P. betae, BSBV+P. betae, BNYVV+BSBV+P. betae and aviruliferous P. betae compared with healthy samples. Mouhanna et al., (2001) stated that by heavier attack of virus, the most severe negative influence are of nematodes and similarly our results indicated these negative effects. Although the virus diseases attack mainly plant leaves, viruses also attacking the roots, like rhizomania, may cause negative effect on cyst nematodes. The number of side roots is increased by rhizomania. So, the negative effects on cyst nematodes could be due to the root quality (Mouhanna et al., 2001). Consequently, it is necessary to evaluate the result of this study with artificial certain number inoculations of all viruses, their vector P. betae and SBN in order to confirm the effects of all to each other and also sugar beet plants.

Fig. 2 (A-D): A. Juvenile in early stages of SBN in root tissues of bait plant; B. Juveniles in late stage of SBN in root tissues of bait plant; C. Young mature female of SBN around roots of bait plant; D. P. betae cystosori in bait plant root.

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