Quantitative methods for assessment of the impact of different crops on the inoculum density of Rhizoctonia solani AG2-2IIIB in soil - PDF

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DOI /s Quantitative methods for assessment of the impact of different crops on the inoculum density of Rhizoctonia solani AG2-2IIIB in soil Barbara Boine & Anne-Catherine Renner
DOI /s Quantitative methods for assessment of the impact of different crops on the inoculum density of Rhizoctonia solani AG2-2IIIB in soil Barbara Boine & Anne-Catherine Renner & Michael Zellner & Jan Nechwatal Accepted: 1 August 2014 # Koninklijke Nederlandse Planteziektenkundige Vereniging 2014 Abstract Rhizoctonia solani AG2-2IIIB is the causal agent of late crown and root rot in sugar beet. In a 4-year field study we analyzed the impact of different plant residue management systems of sugar beet and maize as well as of growing wheat (non-host) and different maize varieties on the soil inoculum density of R. solani. Sugar beet remains were either tilled or removed from the field; maize was then grown during the two following years and also tilled or removed. The soil inoculum potential of R. solani was studied using three different on- and off-site monitoring systems. A monthly assessment of root damage indices of maize and sugar beet and broad bean as an indicator plant was carried out. In addition, an indirect quantitative real-time PCR assay using quinoa seed baits was developed to analyze field soil samples for R. solani AG2-2 soil concentration at the end of each year. The results show that the non-host wheat as a pre-crop to sugar beet reduced the Rhizoctonia inoculum potential in the soil significantly. Additionally, the incorporation of host plant debris (sugar beet + maize) into the soil increased the Rhizoctonia soil inoculum potential and the incidence of sugar beet rot. Although the maize genotypes susceptibility to R. solani differed, their plant debris did not significantly influence growth and survival of R. solani in the soil. Electronic supplementary material The online version of this article (doi: /s ) contains supplementary material, which is available to authorized users. B. Boine: A. C. Renner : M. Zellner : J. Nechwatal (*) Bavarian State Research Center for Agriculture, Institute for Plant Protection, Lange Point 10, Freising, Germany This work describes methods that allow elucidating the effect of agricultural practice on Rhizoctonia levels in the soil and on disease development in the field. Keywords Quantitative real-time PCR. Late crown and root rot of sugar beet. Indicator plant. Quinoa seed bait. Soilborne fungal pathogen Introduction Rhizoctonia solani AG2-2IIIB is the causal agent of late crown and root rot in sugar beet (Beta vulgaris subsp. vulgaris) and causes considerable yield losses worldwide. Due to R. solani s ubiquitous nature and wide host range, late root and crown rot poses a constant threat to sugar beet production. While the late crown and root rot of sugar beet is caused by a Rhizoctonia complex of different anastomosis groups (AGs) and subgroups (Cubeta and Vilgalys 1997), the AG2-2 subgroup IIIB is the most aggressive type infesting sugar beet roots (Büttner et al. 2002). In Europe, more than 36,000 ha are affected (Garcia et al. 2001). In the United States subgroup AG2-2IV was found being similarly destructive (Engelkes and Windels 1996). In contrast to the USA, chemical disease control of R. solani in sugar beets is not feasible because no agents are registered yet in Germany. Similarly, biocontrol agents against R. solani are not on the market yet. The only measure against the late crown and root rot of sugar beet are Rhizoctonia resistant varieties, even though a yield drag must be taken into account, especially for very new varieties. Also, integrated control strategies like crop rotation and plant residue management are decreasing disease incidences (Anees et al. 2010). However, sorghum, soybean, other edible bean species, and maize are also known to be hosts of R. solani AG2-2IIIB and can increase disease severity in sugar beet when grown as a pre-crop (Rush and Winter 1990; Windelsand Brantner 2004). Although researchers have been studying the genus Rhizoctonia for nearly 200 years (Duggar 1915), the population dynamics of R. solani in soil and how environmental factors are interacting to trigger Rhizoctonia root rots still needs to be elucidated. The recent worldwide increase in cultivation of other R. solani AG2-2 hosts, like maize (Sumner and Bell 1982; Kluth and Varrelmann 2010) or soybean (Liu and Sinclair 1991; Windels and Brantner 2007) is presumed to elevate Rhizoctonia soil inoculum densities. Extreme and unstable weather conditions (e.g. heavy rain falls), likely to increase in future due to global climate change, might amplify Rhizoctonia outbreaks (e.g. Garrett et al. 2011). If extreme weather events coincide with certain local geological morphologies, e.g. heavy rains filling up swales, even low Rhizoctonia soil inoculum densities might be sufficient for triggering Rhizoctonia rot. Besides host plant availability and weather conditions, several other factors seem to influence the Rhizoctonia inoculum potential in the soil: e.g. soil type and structure, crop rotation patterns including Rhizoctonia hosts, plant residue management, tillage practices and microbial soil community (Buhre et al. 2009; Anees et al. 2010). In order to study the dynamics of Rhizoctonia soil inoculum potentials, sensitive and reliable detection and quantification methods are required. While other anastomosis groups (AGs) of R. solani are routinely quantified by PCR-based methods (Lees et al. 2002: AG3; Paulitz and Schroeder 2005: AG8; Sayler and Yang 2007: AG1-1A; Okubara et al. 2008: AG2-1, AG8, AG10), the late root and crown rot of sugar beet is commonly approximated by determining amounts of rotted plants or infested fields (Neate and Schneider 1996). Hence, the disease is only recognized when Rhizoctonia has already spread and manifested itself across the field and yield losses can no longer be prevented. A routine quantification method for R. solani AG2-2 density in soils to forecast the potential for late root and crown rot of sugar beet would therefore be desirable. Here we report on two monitoring systems estimating the R. solani AG2-2 soil inoculum densities during cultivation of sugar beet, maize and winter wheat, a non-host for R. solani AG2-2 which might directly or indirectly prevent inoculum build-up in the soil (e.g. Windels and Brantner 2004; Buhre et al. 2009). Field trials were conducted over a period of 4 years ( ). The first monitoring system was an on-site bio-assay using the indicator plant Vicia faba, commonly known as broad bean, which was grown between the crop rows. Second, a molecular assay using quantitative real-time PCR and quinoa (Chenopodium quinoa) seed baits was established (quinoa-qpcr assay) in order to specifically quantify R. solani AG2-2 in soil. Utilizing these two monitoring systems, two main objectives were addressed. The first objective was to quantify the effect of different pre-crops of sugar beet on the Rhizoctonia soil infestation level and on the disease development in sugar beet. Within this first objective, the question whether plowing organic matter into the soil contributes to a pre-crop effect was also addressed. The second objective was to compare different maize genotypes regarding their impact on the Rhizoctonia soil inoculum level. Material and methods Rhizoctonia isolate and culture conditions The Rhizoctonia solani AG2-2IIIB isolate RS580 was originally isolated from a diseased sugar beet root in northern Germany in RS580 was routinely cultivated on malt extract agar (20 g agar agar, 20 g malt extract powder (both Carl Roth, Karlsruhe, Germany) per litre of distilled water) (MEA) at 23 C for 2 weeks and then stored at 17 C until required. The fungal isolate was sub-cultured every 3 months for culture maintenance. Field trials Inoculum production using barley For field trials for each hectare 2 40 kg of Rhizoctonia inoculum were produced. For this purpose, 2.5 kg of cleaned and dried barley grains were thoroughly soaked in sterilized distilled water overnight and then filled into SacO 2 Microsacs (Mycelia, Belgium). The remaining water was drained carefully and the grain was autoclaved twice. The sterile barley grains were partially dried overnight under sterile conditions, then inoculated with R. solani AG2-2IIIB (isolate RS580). Each Microsac was inoculated with Rhizoctonia mycelium from four agar plate cultures (MEA, Ø 9 cm). For inoculation the agar was chopped into small pieces, transferred into the Microsacs and mixed well. Afterwards the Microsacs were sealed and incubated for 2 weeks at 23 C in the dark. The Microsacs were turned and shaken twice a day to prevent the formation of grain clots. The barley grains were then dried under continuous air flow in an extractor hood (non-sterile) until the weight was steady. Each lot of inoculum was tested for Rhizoctonia growth and potential contamination by placing 10 grains of each lot on a MEA plate for 1 week. Additionally, the inoculum was checked for pathogenicity and virulence using shoots of broad bean (Vicia faba) cultivar Espresso. For each lot five sprouted beans were placed onto MEA plates (one bean per plate) and exposed to two Rhizoctonia infested grains. Lots with highest virulence levels and without contamination were pooled and used for inoculation of field plots. Field trial set-up Field trials were conducted from 2009 until 2012 in a sugar beet growing region in southern Germany (Lower Bavaria, Haardorf, N, E). At the beginning, all plots (plot size approx. 40 m 2 )were inoculated with artificially infested barley (see above) and sown with sugar beet. Three single plots remained uninoculated as a control. At the end of the season in 2009, the field trial was separated into two parts. The first part was called Crop rotation trial (CR-trial) and the second was called Maize variety trial (MV-trial). The CR-trial investigated the impact of growing different pre-crops (maize and winter wheat) of sugar beet on the Rhizoctonia inoculum soil potential (Table 2). In addition, different plant residue management systems were compared. The MV-trial investigated the impact of three different maize varieties on the inoculum potential of R. solani AG2-2IIIB. Experimental designs are explained in detail in Table 1, cultivars and varieties used in the trials are listed in Table 2. The plots of the CR-trial were further split into two sections in While on one section the sugar beet remains were removed after harvest, sugar beet residues of the other section were plowed into the soil in November. These two treatments were called with residue removal (wrr) and without residue removal Table 1 Crop rotation and maize variety field trials, experimental designs Trial name Crop rotation trial (CR-trial) Maize variety trial (MV-trial) 1 st inoculation with 40 kg/ha barley inoculum. 2 nd inoculation with 40 kg/ha barley inoculum Section A: Susceptible sugar beet variety, wrr Section B: Susceptible sugar beet variety, w/orr Susceptible sugar beet variety, w/orr Section A + B: Fully randomized block design with four replicates each of sweet maize, silage maize, and winter wheat 3 varieties of maize: - Banguy - Fabregas - Lacta Section A + B: Fully randomized block design with four replicates each of sweet maize, silage maize, and winter wheat 3 varieties of maize: - Banguy - Fabregas - Lacta Section A + B: Susceptible and resistant sugar beet variety Susceptible and resistant sugar beet variety wrr with sugar beet residue removal, w/orr without sugar beet residue removal Table 2 Plant cultivars and varieties used in the trials Crop plant Variety Trial Source Sugar beet Donella (2009) CR + MV KWS Saat AG, Einbeck, Germany Nauta (2012) CR + MV KWS Saat AG, Einbeck, Germany Belinda (2012) CR + MV KWS Saat AG, Einbeck, Germany Maize Banguy CR + MV KWS Mais GmbH, Einbeck, Germany Lacta MV KWS Mais GmbH, Einbeck, Germany Fabregas MV KWS Mais GmbH, Einbeck, Germany Winter wheat Cubus CR KWS Lochow GmbH, Bergen, Germany Broad bean Espresso CR + MV SAATEN-UNION GmbH, Isernhagen HB, Germany CR crop rotation trial, MV maize variety trial (w/orr), respectively. In 2010 and 2011 maize was grown on two thirds of the plots also being either removed (silage maize) or plowed (grain maize) after harvest in November. Additionally, winter wheat was grown as a non-host of R. solani AG2-2IIIB. In 2012, all plots were sown with both a susceptible and a resistant sugar beet variety. In summary, the following treatment combinations were investigated: 1, wrr silage maize; 2, w/orr silage maize; 3, wrr grain maize; 4, w/orr grain maize; 5, wrr winter wheat (no root damage assessment); 6, w/orr winter wheat (no root damage assessment). In the MV-trial, sugar beet residues were plowed on all plots. The three maize varieties Banguy, Lacta and Fabregas were grown in 2010 and 2011, after and prior to sugar beet cultivation in 2009 and Assessment of root damage due to R. solani Root damage of maize was assessed according to Buddemeyer et al. (2004) once a month from June to September in 2010 and The rating system ranged from 1 (no lesions) to 9 (root dead). Infestation rates of sugar beet were recorded monthly as percent surface area affected using 10 %-steps from June to September in As the resistant beet variety in general showed low levels of disease, only the data of the susceptible variant were used for further comparisons. Root damage on the non-host plant winter wheat was not assessed. Indicator plant assay Broad bean (Vicia faba) cv. Espresso was chosen as an indicator plant for late crown and root rot caused by R. solani AG2-2IIIB based on previous results (data not shown), showing that broad bean is susceptible to the pathogen. For indicator plant assays in the field, bean seeds were sown between plant rows at two different time points each year (end of May and June) from 2010 until Surrounding plants hampering the growth of the beans by shading them were either shortened or removed. Root damage of beans was assessed after 4, 8, 12, and 16 weeks by estimating the percentage of root surface infested in a stepwise manner using 10 %-steps. If necessary, bean roots were washed prior to examination for better assessment of rotted root area. Every assessment included 5 10 replicates. Lab experiments Inoculum production using poppy seeds For lab experiments and creation of qpcr standard curves, 20 g seeds of the common poppy (Papaver somniferum) were mixed with 8 ml water and autoclaved twice before being inoculated with finely chopped agar plugs of two MEA plates fully grown with R. solani isolate RS580. Poppy seeds and R. solani agar pieces were thoroughly mixed and incubated for 2 weeks at 23 C in the dark. Afterwards the R. solani infested poppy seeds were separated from each other using a small spatula and dried under sterile conditions until the weight was steady. The poppy seed inoculum was checked for colonization by R. solani and for contamination by placingitonmeaplatesandincubatingat23 Cfor2days. Quinoa-qPCR assay A molecular assay was used for specific quantification of R. solani AG2-2. The quinoa-qpcr assay was carried out once a year in October using soil samples (six pooled subsamples each plot) taken from the topsoil (5 to 10 cm below soil surface). Samples were homogenized and passed through a 4-mm mesh sieve to remove plant debris. Two subsamples of the pooled soil sample were then filled into two petri dishes (Ø 9 cm) with 50 g soil each. On top of the soil of each petri dish 10 autoclaved quinoa seed (Chenopodium quinoa) baits, as described by Thornton et al. (1999), were evenly distributed. Soil samples were incubated for 4 days at 23 C in the dark. After incubation the Rhizoctonia infested quinoa seeds were transferred into two plastic tubes and stored at 80 C. For quantification of Rhizoctonia inoculum levels, DNA was extracted from the quinoa seeds using the ZR Plant/Seed DNA MiniPrep Kit (Zymo Research Europe GmbH, Freiburg, Germany) according to manufacturer s recommendations. Primer sequences were obtained from Budge et al. (2009), which were specifically designed to the ribosomal internal transcribed spacer (ITS) regions of R. solani AG2-2. Quantitative real-time PCR (qpcr) was performed using the SensiMix SYBR No-ROX Kit (Bioline, Luckenwalde, Germany) according to manufacturer s recommendationsin a CFX96 real time system (Bio-Rad, Munich, Germany). The 25 μl reaction mixture contained 12.5 μl SensiMix (Bioline), 0.3 μm of each primer, 6 μl H 2 O, and 5 μl of extracted DNA. In order to convert Ct values into number of sclerotia (or colony forming units (CFU)) per gram soil, a standard curve was developed. A silty loam soil was collected from a non-inoculated field site (depth of 5 10 cm), sieved through 4-mm mesh and autoclaved three times (each 20 min, 121 C) before airing it thoroughly. To simulate actively growing Rhizoctonia sclerotia, poppy seeds infested with R. solani isolate RS580 were used (see above). Each infested poppy seed represented one sclerotium (CFU). The variation of the DNA content between Rhizoctonia infested poppy seeds was very low (the mean Ct of 5 replicates of g poppy seeds was 21.48±0.95). To create a standard curve, 100 g of autoclaved soil was mixed with different numbers of infested poppy seeds. A quinoa bait assay as described above was then performed with these soil samples to obtain Ct values for known concentrations of CFUs. The formula of the standard curve trend line was used to calculate unknown Rhizoctonia soil densities in CFU per 100 g soil (Fig. S1). Statistical analysis Differences of root damage indices of maize and differences of percentage of infected root surface of broad bean and sugar beet were analyzed using a one-way analysis of variance (ANOVA). Differences between treatment combinations were analyzed using Tukey pairwise comparison. Before the analysis, the normality of the percentage of infected root surface data for each host was tested using a Kolmogorov-Smirnov test. P values smaller than 0.05 were considered statistically significant. SAS 9.2 software (SAS Institute Inc., Cary, NC, USA) and GraphPad Prism 3.00 Software (GraphPad Software, San Diego, CA, USA) were used to perform these calculations. Root damage data of indicator and crop plants were analyzed for best assessment time by using the PROC REG procedure (selection=stepwise) in SAS 9.2. As a result of this selection analysis, root damage assessment data of maize and bean recorded in July and of sugar beet recorded in September were used for final data comparisons. Results Irrespective of the field test set-ups and parameters assessed, the inoculation with Rhizoctonia infested barley seeds was successful. Data originating from the infested plots were clearly distinguishable from those of the control plots and most parameter sets retrieved indicated an elevated Rhizoctonia soil content in the inoculated plots, although some natural infestation was also observed in the controls (data not shown). Crop rotation-trial Figure 1a and b show the root damage assessment of maize separated by treatment combinations 1) to 4) in 2010 and Because maize roots were equally affected by the artificial R. solani inoculum, no differences were determined among treatment combinations studied in 2010 and On the other hand, a slight general decrease in maize root damage from 2010 to 2011 could be observed, possibly due to the dilution of the initial inoculum. However, when sugar beet was grown on all plots in 2012 a treatment effect did become apparent (Fig. 1c): when sugar beet was grown after 2 years of maize cultivation, Rhizoctonia root rot incidence was higher than when grown after Fig. 1 Box plot diagrams of root damage assessments of maize grown in 2010 and 2011 (a+b/left + center) and sugar beet grown in 2012 (c) with different plant residue management systems. Winter wheat was grown in 2010 and 2011 (a+b/right), but root damage was not determined (n.d.). wrr: 2009 sugar beet residues removed from the field, w/orr: 2009 sugar beet residues plowed after harvest. In
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