Journal of Plant Diseases and Protection, 117 (4), 150–155, 2010, ISSN 1861-3829. © Eugen Ulmer KG, Stuttgart

Investigation of the persistence of Beet necrotic yellow vein virus in rootlets of sugar beet during biogas fermentation Untersuchung der Überdauerung des Rübenwurzelbärtigkeits-Virus in Zuckerrübenwurzeln im Biogasfermenter R. Friedrich, D. Kaemmerer & L. Seigner* Bavarian State Research Center for Agriculture, Freising, Germany * Corresponding author, e-mail [email protected] Received 22 January 2010; accepted 26 May 2010

Abstract Rhizomania of sugar beet caused by the Beet necrotic yellow vein virus (BNYVV) and transmitted by Polymyxa betae leads to great losses of yield in sugar beet production worldwide. The virus is multiplying in the sugar beet and its roots are finally enclosed in P. betae sporosori and released into the soil during decomposing. Virus and vector sporosori remain infectious in the soil for years. Setting free sporosori und BNYVV into the environment during sugar production and deposing of contaminated waste implies a phytosanitary risk for spreading rhizomania. Fermentation of plant residues in biogas plants could possibly lead to inactivation of P. betae and/or BNYVV. Within a research project, conducted at the Bavarian State Research Center for Agriculture (LfL), the persistence of different pathogens – including P. betae and BNYVV – during biogas fermentation was studied to evaluate the hygienisation potential of the biogas process and to assess the risk of spreading pathogens with the fermentation residues. To investigate BNYVV tenacity, infected rootlets of sugar beet plantlets were incubated at 38°C and 55°C in 36 l-continuous fermenters up to 36 days and analyzed by ELISA directly after fermentation. In addition, to check the infectivity of the vector/virus complex a bioassay was performed. When fermented at 38°C, BNYVV could be detected by ELISA in the rootlets for up to 36 days, but only during the first 4–8 days of incubation infectivity of virus and/or vector were retained. Compared with incubation at 38°C, incubation at 55°C led to an enhanced degradation of BNYVV and accelerated loss of viral and/or vector infectivity: BNYVV antigen concentration was dramatically reduced and reached a minimum after 3 weeks of incubation. Viral and/or vector infectivity were lost very fast already during the first 4 days of incubation in the fermenter. It can be assumed that biogas fermentation under thermophilic but also mesophilic conditions leads to a relatively fast inactivation of BNYVV and/or its vector P. betae. At least with respect to rhizomania, widely harmless fermentation residues should be produced if the retention time in the fermenter at temperatures between 38°C and 55°C is at least 1 week. But nevertheless, for a full assessment of the phytosanitary risk additional studies are required. Key words: bioassay, BNYVV, ELISA, fermentation, incubation time, Polymyxa betae, rhizomania, temperature effect, vector

können auf den Kulturflächen jahrelang infektiös bleiben. Werden Sporosori und BNYVV während der Zuckerproduktion und bei der Entsorgung kontaminierter Abfälle in die Umwelt freigesetzt, so ergibt sich auch daraus ein phytosanitäres Risiko für die Verbreitung der Rizomania. Eine Möglichkeit der Inaktivierung des Vektors und/oder des BNYVV könnte die Vergärung des Pflanzenmaterials in Biogasanlagen sein. In einem Forschungsprojekt, durchgeführt an der Bayerischen Landesanstalt für Landwirtschaft (LfL), wurde die Persistenz verschiedener Phytopathogene – einschließlich P. betae und BNYVV – während der Biogasfermentation untersucht, um das Hygienisierungspotential des Biogasprozesses und das Risiko einer Verschleppung von Pathogenen mit dem Gärrest abzuschätzen. Um das Überdauerungsvermögen des BNYVV zu analysieren, wurden infizierte Zuckerrübenwurzeln bei 38°C und 55°C in 36 l-Durchflussfermentern über einen Zeitraum von bis zu 36 Tagen inkubiert und direkt nach der Inkubation mittels ELISA getestet. Zusätzlich wurde zur Überprüfung der Infektiosität des Vektors bzw. Virus ein Biotest durchgeführt. BNYVV konnte nach Inkubation im Fermenter bei 38°C bis zum Versuchsende am 36. Tag in den Wurzeln mittels ELISA nachgewiesen werden, aber nur während der ersten 4–8 Tage der Inkubation blieb die Infektiosität des Virus und/oder Vektors erhalten. Im Vergleich zur Fermentation bei 38°C führte eine Inkubation bei 55°C zu einem beschleunigten Abbau von BNYVV und einer rascheren Inaktivierung des Virus und/oder Vektors: Die Konzentration an BNYVV-Antigenen wurde dramatisch reduziert und erreichte nach 3-wöchiger Inkubation ein Minimum, die Virulenz ging rasch bereits während der ersten 4 Tage der Inkubation im Fermenter verloren. Man kann davon ausgehen, dass die Biogasfermentation unter thermophilen, aber auch unter mesophilen Bedingungen zu einer relativ schnellen Inaktivierung des BNYVV und/oder seines Vektors P. betae führt. Zumindest bezüglich Rizomania sollten weitgehend unbedenkliche Gärreste gewonnen werden, wenn die Verweildauer des Substrats im Biogasfermenter bei Temperaturen zwischen 38°C und 55°C mindestens eine Woche beträgt. Dennoch sind zur vollständigen Abschätzung des phytohygienischen Risikos weitere Studien notwendig. Stichwörter: Biotest, BNYVV, ELISA, Fermentation, Inkubationsdauer, Polymyxa betae, Rizomania, Temperatureffekt, Vektor

1

Introduction

Zusammenfassung Die Rizomania der Zuckerrübe, die durch das Beet necrotic yellow vein virus (BNYVV) (Rübenwurzelbärtigkeits-Virus) verursacht und durch den Vektor Polymyxa betae übertragen wird, führt weltweit zu großen Ertragsverlusten. Das Virus, das sich im Rübenkörper und in den Wurzeln vermehrt, gelangt eingeschlossen in P. betae-Sporosori über Pflanzenreste in den Boden. Das Virus und die Sporosori des Vektors

Polymyxa betae (KESKIN 1964), which belongs to the Plasmodiophoraceae, is the soil-borne fungal vector of the Beet necrotic yellow vein virus (BNYVV) (TAMADA 1975), the causal agent of rhizomania of sugar beet. Worldwide, rhizomania is an important problem since infection causes serious losses of yield (SCHOLTEN and LANGE 2000) by a massive proliferation of lateral roots resulting in smaller beets and a reduced sugar content (CIAFARDINI 1991). P. betae itself does not cause sigJ.Plant Dis.Protect. 4/2010

Friedrich et al.: Persistence of BNYVV in sugar beet rootlets during biogas fermentation nificant damage, but it enables the virus to remain infectious within its sporosori (cystosori or resting spore clusters) in the soil for several years (CIAFARDINI 1991). The remaining resting spores in the soil and infected plant residues after harvest provoke the disease outbreak in the following growing season (STACEY et al. 2004). One approach to counteract the yield losses caused by BNYVV is breeding for resistance. By now, rhizomania tolerant cultivars are available and today rhizomania seems not to be a real problem. But growing tolerant cultivars leads to an enrichment of BNYVV in the soil and implies the danger of selecting virus strains with higher pathogenicity or even resistance-breaking strains so that rhizomania could be a problem again in future, perhaps even worse than in the past (KINGSNORTH et al. 2003; KOENIG et al. 2009). For phytosanitary reasons, with respect to spreading pathogens the disposal of infested plant material and waste means a risk. Therefore problems may also arise from setting free sporosori and BNYVV into the environment during sugar production and concomitant waste disposal. Fermentation in biogas plants could offer a chance to sanitize infected plant material. The biogas technology has reached great importance in recent years because it affords the use of renewable sources for energy production. Moreover, it provides the opportunity to utilize the fermentation residue as fertilizer on the fields and to dispose of contaminated plant material. Not many data are available concerning the survival of pathogens in biogas plants so far. If pathogens survive the fermentation process, there is a high risk for spreading them extensively together with the fermentation residue on the field. This could probably lead to an escalating increase of pathogens on production sites, repeated and early infestation of the crop resulting in heavy losses of yield and economic damage for the farmers. To evaluate the hygienisation potential of the biogas fermentation process with regard to different plant pathogens, a research project was started at the Bavarian State Research Center for Agriculture (LfL). Results from this project concerning the persistence of BNYVV in infested sugar beets at meso- and thermophilic conditions in the biogas fermenter are presented here.

2 Materials and methods 2.1 Biogas fermenters and sentinel chambers For the fermenter experiments, 36 l-continuous fermenters at the Institute for Agricultural Engineering and Animal Husbandry (ILT, LfL) were used to analyse the persistence of BNYVV simulating practical fermentation conditions. These digesters were run at mesophilic (approx. 38°C) and thermophilic (approx. 55°C) temperature range with mainly grass and maize silage as substrate. The volumetric loading in the fermenters varied between 0.5 and 2.5 kg dry matter per m3 and day throughout the experiments. To introduce the infected sugar beet rootlets in the fermentation process and to ensure the recovery of this material for further analysis, special carriers (sentinel chambers) made of Duran glass with a volume of 10 ml and a diameter of 2 cm had been developed. In Fig. 1A the details and components of the sentinel chambers are described. The sentinel chambers were closed with filter membranes (pore size 0.8 Pm) at both ends prohibiting Polymyxa betae spores to escape into the digester by simultaneously ensuring the indispensable exchange with the digester content. Two of these carriers were fixed in the carrier holding system (Fig. 1B) and then placed in the fermenter (Fig. 1C–D).

2.2 Cultivation of plants To provide infected plant material and positive controls, 30 sugar beet seeds (Beta vulgaris subsp. vulgaris var. altissima; J.Plant Dis.Protect. 4/2010

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variety “Ricarda”) were sown in rhizomania-infested soil, provided by the Institute for Crop Science and Plant Breeding (IPZ, LfL), in 7 u 7 u 8 cm pots. Negative controls were produced essentially in the same way, but sown in sterilized sand. The plants were grown in the greenhouse at 24°C for 8–10 days till germination and were further cultivated at 21°C during the day (8 a.m. – 8 p.m.) and 18°C during the night. Supplementing light was given between 6 a.m. and 20 p.m. when natural radiation intensity was between 10 to 60 klux. The whole cultivation from sowing till harvest lasted 6–8 weeks. During growth, plants were fertilized three times with Wuxal® (2 ml l–1 water; Aglukon Spezialdünger GmbH, Düsseldorf, Germany).

2.3 Plant material, incubation in the biogas fermenter and sampling 6–8 weeks old sugar beet rootlets were used for the fermenter experiments. After harvesting, the adherent soil was thoroughly washed off from the rootlets and the remaining water was gently dabbed off with a paper towel. To check the BNYVV infection status, root material was analyzed via ELISA (see 2.4). Additionally, roots were examined microscopically for resting spores of P. betae. Infected root material (2 g, fresh weight) was introduced into each sentinel chamber which was then filled up completely with digester content obtained from the fermenter used for the subsequent experiment. The sentinel chamber was closed and – in most cases – together with a second sentinel chamber fixed in the carrier holding system as shown in Fig. 1. Sentinel chambers were incubated up to a maximum of 36 days at 38°C and 55°C. Two experiments with different numbers of sentinel chambers and fermenters covering different incubation periods were performed. In the first experiment, a total of 16 sentinel chambers were incubated in 12 different fermenters for 4–22 days. In the second experiment, 18 sentinel chambers were incubated in nine different fermenters for 21–36 days. For regaining the sample material after the predefined incubation periods, the sentinel chambers were removed from digesters. For the first experiment 1–5 sentinel chambers and for the second experiment 1–4 sentinel chambers were taken at each sampling date from the 38°C and 55°C digesters, respectively. Mostly, at each sampling date at least the two sentinel chambers of one fermenter were removed, but in four cases due to a failure of the digester only one sentinel chamber was available. The content of one sentinel chamber was treated as one sample. Each sample was tested by ELISA (see 2.4) and bioassay (see 2.5).

2.4 ELISA analysis ELISA analysis was performed to test the sugar beet rootlets for BNYVV prior to the fermenter experiments, after incubation in the fermenter and after bioassay (see 2.5). Root material harvested from pots after plant cultivation or from sentinel chambers was rinsed with water until the water runs clear. Then, if the rootlets were taken from the sentinel chambers, they were shortly rinsed with 80% ethanol and again with water. Afterwards, the rootlets were gently dried on a paper towel. Samples (500 mg fresh weight) of root material were weighed out in a homogenization bag (10 u 10 cm, Zefa, Harthausen, Germany) and kept at –80°C until ELISA was performed. Rootlets from non-infected sugar beet plants were used as negative controls, rootlets from infected plants as positive controls (see 2.2). Anti-BNYVV-Ig G and Ig G-alkaline phosphate conjugate for ELISA were purchased from Loewe Biochemica GmbH (Sauerlach, Germany). Buffers, Ig G and conjugate dilutions as well as substrate solution were prepared as recommended by Loewe. Prior to ELISA, the wells of the microtiter plates (96 Well Immuno Plates, F96 MaxiSorp;

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A

C

B

D

Fig. 1: A) Components of a sentinel chamber: end caps with opening, gaskets, carrier plates, cellulose acetate membrane filters with 0.8 Pm pore size (left to right) and Duran glass cylinder. The sample material and substrate from the fermenter are placed within the glass cylinder before the sentinel chamber is assembled like shown (right). B) Two sentinel chambers are fixed with cable ties in the carrier system. C) Schematic representation: two sentinel chambers placed in the holder of the fermenter like indicated by the arrow. D) A series of 36 l-continuous fermenters at the ILT.

Nunc GmbH & Co. KG, Langenselbold, Germany) were coated with 100 Pl Ig G solution and afterwards washed six times with wash buffer. The frozen plant material was homogenized on ice in cold sample buffer (1:10 w/v). The extract was further diluted 1:2 with sample buffer. After centrifugation (1,000 u g, 1 min), the clarified supernatant (100 Pl) was pipetted into the wells of the microtiter plate which was incubated over night at 4°C and washed as described above. Ninety Pl of diluted conjugate was added to each well, incubated for 4 h at 37°C and again wells were washed six times. Finally, substrate solution (80 Pl) was added to each well. Plates were incubated at 22°C in the dark. Extinction values measured at 405 nm against sample buffer were taken after 45 min, 2 h and 4 h with an ELISA reader (Tecan SunriseTM, Tecan Austria GmbH, Grödig/Salzburg, Austria). Samples with an extinction value exceeding 250 mA after 4 h were considered positive.

2.5 Bioassay For checking not only the presence of BNYVV in the roots after incubation in the fermenter, but also for controlling the infectivity of the virus/vector complex, the roots – in parallel to ELISA – were tested by bioassay. For this purpose, roots were taken out of the sentinel chamber, washed, dried, weighed out as described above and stored at –80°C until ELISA was carried out (see 2.4). The remaining portions of roots (about 1.5 g) of each sample were collected in small nylon bags and introduced into pots which were already filled at one third of their volume with mould. The bag was covered first with 1/3 mould, second with a 2–3 cm layer of perlite and third with 0.5–1 cm sterilized sand. On this layer, 30 sugar beet seeds (see 2.2) were spread and covered with perlite and sand. Finally, the pots were watered with 0.15% (v/v) Previcur N solution (Hoechst-Schering AgrEvo GmbH, Düsseldorf, GermaJ.Plant Dis.Protect. 4/2010

Friedrich et al.: Persistence of BNYVV in sugar beet rootlets during biogas fermentation ny). After 6 weeks of cultivation, the rootlets of the small sugar beet bait plants were analyzed with ELISA for BNYVV (see 2.4).

2.6 Data analysis Due to technical reasons, the overall sample number was limited and the number of samples per sampling were varying. Thus, no comprehensive statistical analysis was performed with the data gained within our experiments. For comparison of ELISA values obtained at the different sampling dates, median values were calculated from the samples (corresponding to different sentinel chambers) taken at 38°C and 55°C. In addition, in the diagrams maximal and minimal values of ELISA absorbance values are given.

3

Results

BNYVV infected rootlets of sugar beet plants harboured in sentinel chambers were incubated at 38°C and 55°C up to 36 days in continuous biogas fermenters to study the hygienisation capacity of biogas fermentation. Samples were taken after different times of incubation and ELISA analysis was performed to prove whether BNYVV was still present in root material after biogas fermentation. Two experiments were performed with a total of 34 samples: in the first experiment 1–5 samples (= 1–5 sentinel chambers) per sampling date were taken from the 38°C-fermenter and 55°C-fermenter; 1–4 samples (= 1–4 sentinel chambers) were taken in experiment 2. In Fig. 2 and 3 results from both experiments are summarized. As shown in Fig. 2, BNYVV was detectable in all root samples incubated at 38°C taken throughout the whole experiment from day 4 till day 36 of fermentation. ELISA values of the fermenter samples showed a tendency to decrease during the 38°C-incubation, but in each case they were undoubtedly positive and more or less in the range of the positive control even at the last sampling date, i.e. after 36 days of incubation. Concerning the different digester temperatures, ELISA values of the samples incubated at 55°C were obviously lower than those gained after incubation at 38°C (Fig. 2). This was already true

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for the first sampling after 4 days of incubation when there was an approximately five-fold difference between the absorbance values. After an incubation of about 3 weeks at 55°C, ELISA values reached a minimum which corresponds almost to the threshold set for discrimination of “positive” and “negative” (see 2.4) in our system. This indicates that in contrast to the 38°C-incubation, incubation at 55°C provokes a drastic reduction in BNYVV antigen concentration. Detection of BNYVV in digester samples directly after biogas fermentation does not stringently imply that BNYVV and its vector P. betae maintained their virulence over 5 weeks of incubation at 38°C and 3 weeks at 55°C, respectively. ELISA only proves the presence of the BNYVV antigen detected by the Ig G applied. Therefore a bioassay was performed with the root samples after incubation in the fermenter. The roots harvested from these bioassay bait plants were analysed via ELISA. As shown in Fig. 3, the results differ clearly from the ELISA analysis immediately after fermenter incubation: only in two of four samples incubated at 38°C for 4 days the ELISA values were positive and even higher than those of the positive controls, whereas in the other samples taken at the same time and in all samples taken later on during the experiment BNYVV could not be detected irregardless of the fermentation temperature. Thus, it can be concluded that BNYVV and P. betae kept their ability to infect bait plants – to a certain extent – when fermented at 38°C during the first 4 days of incubation. It can be further assumed that at least the vector or the virus or possibly both lost their infectivity completely during the first 8 days of biogas fermentation under mesophilic conditions. In contrast to 38°C, incubation at 55°C lead to a complete loss of infectivity already during the first 4 days of fermentation.

4 Discussion As mentioned in the introduction, phytosanitary problems and the risk for setting new infections are associated with BNYVV or BNYVV carrying P. betae spores released into the environment in the course of sugar beet processing and with the resulting waste. In literature, many studies can be found investigating the persistence of P. betae and BNYVV in relation to different temperatures, moisture content and oxygen avail-

Fig. 2: ELISA absorbance values (mA) for BNYVV in rootlets of sugar beet plantlets directly after incubation for different times (1–36 days) in the biogas fermenter at 38°C and 55°C, respectively. A summary of the results of experiment 1 (4–22 days) and experiment 2 (21–36 days) resulting from a total of 34 samples is presented. Medians of blankcorrected ELISA absorbance values (n = 1–5 in experiment 1, n = 1–4 in experiment 2) are shown for each incubation time and both temperatures. Maxima and minima of ELISA values are given as vertical bars except for the days 4/55°C, 8 and 22/55°C, where only one sentinel chamber (= one sample) was available. There is no result for 16 days/55°C because of a breakdown of the fermenter. KK: infected roots frozen at –80°C without incubation in the biogas fermenter used as positive controls for ELISA. J.Plant Dis.Protect. 4/2010

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Friedrich et al.: Persistence of BNYVV in sugar beet rootlets during biogas fermentation Fig. 3: ELISA absorbance values (mA) for BNYVV in rootlets of sugar beet bait plants after bioassay. Bioassays were performed with rootlets of sugar beet plantlets incubated for different times (1–36 days) in the biogas fermenter at 38°C and 55°C, respectively. A summary of the results of experiment 1 (4–22 days) and experiment 2 (21–36 days) resulting from a total of 34 samples is presented. Medians, maxima and minima of ELISA values are given comparable to Fig. 2. As two samples taken after a 4-day incubation at 38°C were clearly positive and two samples were negative, medians of the absorbance values of the positive (4P) and negative samples (4N) are displayed separately. There is no result for 16 days/55°C because of a breakdown of the fermenter. KK: infected roots not incubated in the biogas fermenter used as positive controls for the bioassays.

ability. Moreover, studies have been published dealing with the effect of composting and compost leachate, thermal treatment and oxygen availability on the survival and persistence of P. betae and BNYVV, respectively (NISHINOME et al. 1996; VAN RIJN and TERMORSHUIZEN 2007). Especially temperature seems to have a major impact, moisture and anaerobic conditions contribute to a decreased survival. In the context of rhizomania, it should be kept in mind when pathogen inactivation is discussed that there are two different targets, the virus and its vector. ANONYMOUS (1977) states the thermal inactivation point of BNYVV between 70°C and 75°C, but at room temperature crude juice remains infectious for at least 7 days. ABE (1987) indicates an incubation of 10 min at 60°C at moist conditions to be sufficient to eradicate the fungal vector P. betae. DICKENS et al. (1991) demonstrated that a temperature of 70°C kept at least for 30 min is sufficient to kill all P. betae resting spores. Taking into account all these findings, it can be assumed that P. betae as well as BNYVV are destroyed when exposed to temperatures higher than 70°C. Our results show that anaerobic fermentation in a biogas digester leads to inactivation of BNYVV and/or its vector even under lower temperature conditions, i.e. within 4–8 days at 38°C and within 4 days at 55°C. Applying our testing regime, we cannot discriminate between an inactivation of BNYVV and its vector P. betae. Our results may indicate that inactivation at 38°C is probably mainly targeted against the vector as BNYVV could be detected by ELISA in all the root samples taken immediately after the 38°C-incubation in the fermenter and the ELISA absorbance values were fairly high during the whole experiment, i.e. up to 36 days. In contrast to this, the bioassay gave negative results already after 4–8 days of incubation at 38°C which might suggest vector inactivation during this early phase. But as mentioned above, positive ELISA results only demonstrate the presence of the BNYVV antigen and do not prove the infectivity of the virus. Differences between ELISA detectability of the virus, viral infectivity and vector survival during drying and composting were recently described for Melon necrotic spot virus and its vector Olpidium bornavanus by AGUILAR et al. (2010). Thus in our studies, despite the comparatively prolonged persistence of BNYVV antigen, a loss of viral infectivity already during the first 8 days of incubation at 38°C cannot be excluded. Compared with incubation at 38°C, temperatures of about 55°C lead to

an accelerated degradation of viral antigen and inactivation of the vector and/or BNYVV resulting in the rapid and complete loss of virulence within the first 4 days of biogas fermentation. This demonstrates clearly the negative effect of elevated temperatures on the persistence of the pathogens. The inactivation of the virus/vector complex in the biogas fermenter at temperatures clearly below 70°C gives strong evidence that during fermentation the biochemical and physical processes in the surrounding milieu are additional factors that strongly affect the persistence of the virus and/or the survival of P. betae. Earlier studies showed that the inactivation of P. betae in compost/compost leachate happens at lower temperatures compared to incubation in water (VAN RIJN and TERMORSHUIZEN 2007). TERMORSHUIZEN et al. (2003) and TURNER et al. (1983) demonstrated that different pathogens (e.g. Fusarium oxysporum f. sp. dinathi, Corynebacteriummichiganense, Globoderapallida) can already be inactivated at mesophilic temperatures around 35–37°C when incubated in an anaerobic digester. The inactivation in the biogas fermenter is likely to be induced by synergistic detrimental effects: elevated temperature, anaerobiosis, microbial antagonism and proteolytic degradation of the pathogen by the microflora, low pH-value due to acid production, harmful decomposition products and escharotic substances released during fermentation (BOLLEN and VOLKER 1996; FRAUZ et al. 2006). Looking at the data presented here and looking at the results published by FRIEDRICH et al. 2009, it can be concluded that not only thermophilic but also mesophilic temperature conditions around 38°C are sufficient in inactivating several plant pathogens if combined with anaerobic fermentation and if a defined minimal retention time in the fermenter is kept. This minimal retention time varies between pathogens (FRIEDRICH et al. 2009 and 2010). For BNYVV and P. betae, the pathogen-vector complex studied here, a retention time of at least 8 days at 38°C lead to hygienisation. Thus, it can be assumed that with regard to rhizomania also under practical conditions in a continuous biogas fermenter – where the average retention time for the substrate is 15–40 days in general and 30–40 days in mesophilic biogas plants (ANONYMOUS 2007) – harmless fermenter residues are produced. This is also true for some other but not for all pathogens as shown by FRIEDRICH et al. (2009 and 2010). According to our results obtained under experimental conditions, biogas fermentation – which is a profitable and susJ.Plant Dis.Protect. 4/2010

Friedrich et al.: Persistence of BNYVV in sugar beet rootlets during biogas fermentation tainable possibility for energy production – leads to hygienisation of BNYVV contaminated plant material and waste under thermophilic but also under mesophilic conditions around 38°C. Our findings are of special importance as a study from the Institute for Rural Structural Development, Business Management and Agroinformatics (ILB) of the LfL conducted in 2006 arrived at the conclusion that approximately 75% of biogas plants in Bavaria are run at mesothermal conditions between 36 and 45°C. Only about 10% of the biogas plants work at 46–50°C and 10% under really thermophilic conditions between 51°C and 60°C. In practice, mesothermal fermentation encompasses the range from 30–42°C (ANONYMOUS 2007). A phytohygienic risk with respect to BNYVV cannot be excluded when fermenters are run at temperatures below 38°C because our investigations did not include lower temperatures. Therefore, in further investigations the temperature range should be enlarged to allow a full assessment of the hygienisation potential even at lower temperatures. In this context, also the possible influence of varying substrate compositions and higher load of infested plant material should be analyzed. Finally, it is to be emphasised that our results do not allow for a complete estimation of the risk for spreading rhizomania by fermenter residues as the statistical basis is small and the investigations were performed under artificial conditions more or less simulating practical biogas fermentation. For this purpose, in general a continuous and comprehensive monitoring of biogas plants with respect to plant pathogens would be of great value.

Acknowledgements We thank all LfL-colleagues involved in this project: Margaretha Kappen, Christine Huber, Margarete Kistler, Dorothea Köhler and especially Tim Nerbas from the Institute for Plant Protection (IPS) for their valuable assistance, the colleagues from the Institute for Agricultural Engineering and Animal Husbandry (ILT) for the allocation of the fermenters and the colleagues from the Institute for Crop Science and Plant Breeding (IPZ) for providing BNYVV contaminated soil. Finally, we gratefully acknowledge financial support of the project from the Bavarian State Ministry for Food, Agriculture and Forestry (StMELF).

References ABE, H., 1987: Studies on the ecology and control of Polymyxa betae Keskin, as a fungal vector of the causal virus (Beet necrotic yellow vein virus) of rhizomania disease of sugar beet. Rep. Hokkaido Prefect. Agric. Exp. Stn. 60, 1-99. AGUILAR, M.I., M.L. GUIRADO, J.M. MELERO-VARA, J. GÓMEZ, 2010: Efficacy of composting infected plant residues in reducing the viability of Pepper mild mottle virus, Melon necrotic spot virus and its vector, the soil-borne fungus Olpidium bornavanus. Crop Prot. 29, 342-348. ANONYMOUS, 1977: Composition and structure of Beet necrotic yellow vein virus. J. Gen. Virol. 35, 397-401. ANONYMOUS, 2007: Biogashandbuch Bayern – Materialienband – Kap. 1.1 – 1.5, Bayerisches Landesamt für Umwelt, Augsburg, Germany. http://www.lfu.bayern.de/abfall/fachinformationen/biogashandbuch/index.htm, May 2010. BOLLEN, G.J., D. VOLKER, 1996: Phytohygienic aspects of composting. In: de Bertoldi, M., P. Sequi, B. Lemmes, T. Papi: The Science of Composting 1, 223-246. Blackie Academic & Professional, London, United Kingdom.

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