Evaluation of Rhizoctonia Zeae As a Potential Biological Control Option for Fungal Root Diseases of Sugar Beet K M

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln

Panhandle Research and Extension Center Agricultural Research Division of IANR

2015 Evaluation of Rhizoctonia zeae as a potential biological control option for fungal root diseases of sugar beet K M. Webb Sugar Beet Research Unit, USDA-ARS, [email protected]

R. M. Harveson Panhandle Research and Extension Center, University of Nebraska, [email protected]

M S. West Plains Area Office, USDA-ARS

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Webb, K M.; Harveson, R. M.; and West, M S., "Evaluation of Rhizoctonia zeae as a potential biological control option for fungal root diseases of sugar beet" (2015). Panhandle Research and Extension Center. 73. http://digitalcommons.unl.edu/panhandleresext/73

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RESEARCH ARTICLE Evaluation of Rhizoctonia zeae as a potential biological control option for fungal root diseases of sugar beet K.M. Webb1, R.M. Harveson2&M.S.West3

1 Sugar Beet Research Unit, USDA-ARS, Fort Collins, CO, USA 2 Panhandle Research and Extension Center, University of Nebraska, Scottsbluff, NE, USA 3 Plains Area Ofﬁce, USDA-ARS, Fort Collins, CO, USA

Keywords Abstract Antagonistic fungi; Beta vulgaris; soilborne pathogens; sugarbeet. Several common root diseases routinely damage sugar beet in Nebraska and other production areas of the Central High Plains, and it is becoming more Correspondence: common to find fields infested simultaneously with multiple pathogens. Owing K.M. Webb, Sugar Beet Research Unit, to the shortage of available fungicides for economic management of soilborne USDA-ARS, Fort Collins, CO 80526, USA. Email: [email protected] diseases, alternative techniques such as biological control are increasingly being sought for disease management. Over the last several years, unidentified, Received: 23 June 2014; revised version sterile fungi have been isolated in conjunction with pathogens from infected accepted: 7 February 2015. sugar beet roots and seedlings. At least two promising isolates have been doi:10.1111/aab.12210 identified from in vitro assays that inhibit the radial growth of multiple sugar beet root pathogens, including Rhizoctonia solani, Fusarium oxysporum f. sp. betae, Phoma betae and Pythium aphanidermatum. Based on morphological and molecular characterisation, two isolates, ‘Hall’ and ‘R47’, were putatively identified as Rhizoctonia zeae. In vitro pathogenicity testing indicated that these isolates were not pathogens of sugar beet. Both isolates were compared with the well-established biological control fungus Laetisaria arvalis and tested as potential treatments in a field naturally infested with multiple sugar beet root diseases. Data indicated that these fungi provided some level of protection against a complex of soilborne diseases and suggest that these isolates could have a potential fit in an integrative management strategy for several sugar beet root diseases.

Introduction pathogens can also be found occurring simultaneously causing a generalised root rot disease complex (Harve- Soilborne pathogenic fungi and fungal-like organisms are son & Rush, 2002; Harveson, 2007), and all of these soil- responsible for numerous economically important root borne fungi or fungal-like organisms are known to cause diseases of sugar beet (Beta vulgaris L.) and are major fac- seedling diseases in sugar beet (Cooke & Scott, 1993; tors limiting yields in many production regions of the Harveson et al., 2009). world (Cooke & Scott, 1993; Harveson et al., 2009). These Root rot diseases are more difﬁcult to manage than diseases may include Rhizoctonia damping-off and Rhi- many foliar diseases because they often are not detected zoctonia crown and root rot caused by Rhizoctonia solani before substantial damage has already occurred (Harve- Kühn (Herr, 1996), Fusarium yellows caused primarily by son & Rush, 2002; Harveson et al., 2002a). Furthermore, Fusarium oxysporum Schlechtend.:Fr. f. sp. betae (Stewart) the lack of fungicide options for many members of W. C. Snyder & H. N. Hans (Stewart, 1931; Ruppel, 1991), this root disease complex and the difﬁculty in direct- Phoma root rot caused by Phoma betae A. B. Frank (Bugbee ing fungicides to make contact with target pathogens & El-Nashaar, 1983; Harveson, et al., 2009), and Pythium in the soil make economic control a challenging task root rot caused by Pythium aphanidermatum (Edson) Fitzp. (Harveson et al., 2009). Thus, other techniques for dis- (Kreutzer & Durrell, 1938; Harveson et al., 2009). These ease management are being investigated, including the

Ann Appl Biol (2015) 1 © 2015 Association of Applied Biologists Identification and characterisation of BCA fungi to control sugar beet pathogens K.M. Webb et al. use of resistant cultivars and cultural practices such as & Kimbrough, 2001; Harveson et al., 2002b), P. betae, early planting (Kirk et al, 2008; Harveson et al., 2009), (Zachow et al., 2008) and Pythium spp. (Hoch & Abawi, improved irrigation practices (Harveson & Rush, 2002) 1979; McQuilken et al., 2001; Zachow et al., 2008). Iden- and biological control (Georgakopoulos et al., 2002; tifying potential antagonistic fungi that are found in Collins & Jacobsen, 2003; Galletti et al., 2008). association with sugar beet may lead to new biocontrol Biological control is one form of disease management strategies for managing root diseases. that has been extensively investigated for controlling soil- The objectives of this research were to (a) identify a borne root pathogens, in part due to a global move- group of unknown sterile fungi found occurring natu- ment toward more environmentally sound approaches rally in Nebraska soils in association with sugar beet root for managing pests and diseases (Lewis & Papavizas, 1991; infections and (b) evaluate these sterile fungi as poten- Chet & Inbar, 1994; Alabouvette et al., 2009). Addition- tial BCAs for sugar beet root diseases in field assays. In ally, many biological control agents (BCAs) or antagonis- preliminary studies, these unknown fungi had shown tic microbes can be active against multiple fungal taxa an ability to inhibit other sugar beet pathogens from [reviewed in the study by Chet & Inbar (1994); Fravel multiple taxa including oomycetes, deuteromycetes and (2005); Yang et al. (2009)], whereas fungicides are not basidiomycetes (R.M. Harveson, personal communica- available or may require varied modes of action for man- tion). During in vitro laboratory assays, we tested two of aging all soilborne fungi. For example, foliar fungicides these unknown fungi on their ability to inhibit known are not generally used for controlling field crop root dis- sugar beet pathogens. The most promising of these fungi eases; however, some are registered and quite effective were then tested as potential biocontrol candidates in field for managing Rhizoctonia crown and root rot, includ- studies from 2006 through 2009 and compared with Laeti- ing Quadris (azoxystrobin – FRAC code 11) and Priaxor saria arvalis Burdsall, an established biological control fun- (fluxapyroxad – FRAC code 7 + pyraclostrobin – FRAC gus (Burdsall et al., 1980; Odvody et al., 1980). code 11). Seed treatment fungicides are registered for Pythium spp. and Phoma spp., and these vary with different modes of action (i.e. Hymexazol – FRAC 32 and Materials and methods Thiram – FRAC M3). However, no effective fungicide Isolation from field soils/plants and identification options are currently available for managing Fusarium yellows (Mueller et al., 2013). Various biocontrol agents Candidate fungi for biocontrol were originally discovered have been recognised for decades as potentially control- and isolated from infected sugar beet seedlings that had ling plant disease by reducing pathogen inoculum or been planted into soil samples as part of a diagnostic a pathogen’s disease-producing capacity by the action service for Western Sugar Cooperative sugar beet pro- of one or more mechanisms (Cook & Baker, 1983). ducers (R.M. Harveson, data not shown). The procedure However, the successful establishment of biological con- was used to estimate pathogen populations in production trol strategies in practical applications has been difficult soils prior to planting by conducting a seedling disease to implement (Lewis & Papavizas, 1991; Fravel 2005; assay in the greenhouse (Harveson, 2007; Harveson et al., Xu et al., 2011). Concerns with the safety of biocon- 2014). Briefly, this entailed collecting soil samples from trol agents, in particular on non-target hosts (Louda production fields which were to be planted to sugar beets et al., 2003), complicate the implementation and the use the following year and using these bulked sub-samples of biocontrol agents. Therefore, effective management (obtained from the upper 10- to 15-cm depth) from mul- depends not only on associated plant–microbial interac- tiple locations of the field. All soil samples were col- tions, but also on the ecological fitness of the BCA itself lected in the same manner as those taken for pre-plant (Alabouvette et al., 2009; Xu et al., 2011), as well as on fertility analysis. Typically this included 10–12 subsam- other pragmatic issues such as product formulation, deliv- ples 10 ha−1, with a final volume of at least 1 L per field ery system(s) and field efficacy (reviewed by Lewis & (Harveson et al., 2014). Soil samples were planted with Papavizas, 1991). ‘Monohikari’ which is a sugar beet cultivar suscepti- The use of antagonistic fungal mycoparasites for ble to most soilborne fungal pathogens (Nagata et al., biological control of a soilborne pathogen was first 1991; Harveson et al., 2002a, 2014; Stump et al., 2004) demonstrated by Weindling (1932) on R. solani in citrus and grown for 4 weeks in the greenhouse (maintained seedlings. Since then, numerous cases of antagonistic at ambient air temperatures of 23–28∘C). After emer- fungi have been reported to be effective in several crops gence, symptomatic sugar beet seedlings were harvested against R. solani (Allen et al., 1985; Brewer & Larkin, (Harveson, 2007), plated onto one-half strength acidified 2005; Zachow et al., 2008, 2010), F. oxysporum (Toussoun, potato dextrose agar (1/2APDA; Fisher Scientific, Atlanta, 1975; Alabouvette, 1990; Larkin et al., 1996; Harveson GA, USA), and incubated at room temperature (25–28∘C)

(Sneh et al., 1991). Fungal isolate(s) were then identified 50 mL potato dextrose broth (Becton, Dickinson and Co., based on characteristic mycelial growth emerging from Sparks, MD, USA) with a 7-mm diameter mycelial plug infected hypocotyls (Harveson et al., 2014). from a working PDA culture as described above. Cultures During the course of this project, a number of unrecog- were grown in the dark for 5 days at 25∘C on a rotary nised fungi were found interspersed with cultures of shaker at 100 rpm. Mycelial masses were collected by fil- known sugar beet pathogens which in turn had been iso- tering through double-layered sterile cheese cloth, rinsed lated from diseased sugar beet seedlings. These unknown with de-ionised water, and then lyophilised at −50∘Cfor fungi were isolated and separated by transferring hyphal 48 h. Lyophilised tissue was ground into a fine powder tips to 1/2APDA for purification and further morphologi- using a spatula, and DNA extracted using the Easy-DNA cal identification was performed as described by Sneh et al. extraction kit (Invitrogen, Carlsbad, CA, USA) using the (1991). Morphological characteristics (including sterile manufacturer’s protocol for small amounts of plant tis- hyphal growth and small reddish-brown sclerotia that sues. DNA extractions were performed twice for each iso- ramified throughout media plates) were observed and late. used to identify and distinguish each potential antago- Polymerase chain reaction (PCR) amplification of the nistic fungus (R.M. Harveson, data not shown). To assist internal transcribed spacer (ITS) region was performed in isolate identification, selected isolates were tested for in a total volume of 25 μL by adding 0.5 μL of Fermentas the number of nuclei in the hyphae using the staining Taq polymerase (Glen Burnie, MD, USA), 2.5 μLofthe technique as described by Burpee et al. (1978). Prelimi- manufacturer’s taq buffer, 3.5 μLof25mMMgCl2,3μL nary investigations of these unknown fungi uncovered a of 2 mM deoxynucleotide trisphosphate (dNTPs), 0.3 μL potentially antagonistic character, based on paired plating of each primer, 5 μL of DNA and 10 μL of nanopure experiments with several pathogens of sugar beet includ- water. Amplification of the ITS region was performed ing R. solani, P.betae, and P.aphanidermatum, and the obser- with the ITS1 and ITS4 primers previously described by vation of inhibition of growth of the pathogens by the White et al. (1990). PCR amplifications were performed unknown fungi (data not shown). in a Mastercyler gradient thermo cycler (Eppendorf, Hamburg, Germany) with the following conditions: one Isolate storage and maintenance cycle of 95∘C for 5 min followed by 33 cycles of 94∘Cfor 1 min, 55∘C target-melting temperatures (Tm) for 1 min, All isolates in this study were stored on filter papers at and an extension cycle of 72∘C for 2 min, followed by −20∘C using modified protocols described by Peever & final extension cycle of 72∘C for 5 min. PCR products Milgroom (1992) and Fong et al. (2000). Working cul- were held at 4∘C until they could be removed from the tures were maintained on potato dextrose agar (PDA; thermo cycler. All PCR amplifications were repeated Becton, Dickinson and Co., Sparks, MD, USA), grown at least twice. PCR amplicons were first visualised on at 25∘C and 16-h photoperiod for 7 days, then main- a 1.5% agarose gel stained with EZ-Vision DNA Dye tained at room temperature, and transferred a maxi- (Amresco, Solon, OH, USA) and viewed with a GelDoc mum of two times using established protocols (Parme- system (Bio-Rad, Hercules, CA, USA) and if a single band ter, 1970; Leslie & Summerell, 2006). Isolates included was present, amplicons were purified using the Epoch two unknown potential antagonistic fungi (‘R47’ and Genecatch PCR Clean up kit (Sugarland, TX, USA). ‘Hall’), the known biological control fungus L. arvalis PCR amplicons were sequenced in both directions with (‘Laet’) originally obtained from M.G. Boosalis (Burdsall primers used in amplification by Eurofins MWG/Operon et al., 1980), as well as two isolates each of F. oxyspo- (Huntsville, AL, USA). Consensus sequences for each rum f. sp. betae (‘F19’ and ‘Fob220a’) (Hill et al., 2011), isolate were generated and edited using Geneious ver- P. aphanidermatum (‘Py54’ and ‘WE41’) (R.M. Harveson, sion 6.1.6 (Kearse et al., 2012) software. All sequence data not shown), Phoma spp. (‘Darnell’ and ‘Phoma’) data for each isolate have been deposited into Gen- (R.M. Harveson, data not shown) and R. solani AG2-2 IIIB Bank (http://www.ncbi.nlm.nih.gov/genbank) under (‘R-9’ and ‘R-1’) (Hecker & Ruppel, 1975; L. Panella & the submission number #1710040. The consensus L.E. Hanson, personal communication), all of which are sequence from each isolate was taxonomically iden- known to be pathogenic to sugar beet. tified from the ITS region and the most similar sequence in the GenBank nonredundant (nr) nucleotide database Isolate identification by polymerase chain reaction (http://blast.ncbi.nlm.nih.gov/Blast.cgi) using the Basic amplification and phylogenetic analysis of internal Local Alignment Search Tool (BLASTn). transcribed spacer genomic region The consensus ITS sequence for each unknown iso- Liquid cultures of the two unknown fungi (R47 and Hall) late (R47 and Hall) was used to perform a nucleotide were initiated by inoculating a 250-mL flask containing search in GenBank to identify similar ITS sequences

(using the same primer sequences) from Rhizoctonia zeae, prepared, with four plates of each treatment being placed R. oryzae, and L. arvalis (as an outgroup control) for use into one of three incubators set at 20∘C, 25∘Cand30∘C, in further phylogenetic analysis. BLASTn was used to respectively, and all plates were incubated in the dark. The search sequences within each species individually with radial growth of each isolate on each plate was measured 62 R. zeae,22R. orzyae,and5L. arvalis sequences iden- at 3, 5, 7, 9, and 11 days after plating (dap). The entire tified. All sequences from this search were downloaded experiment was repeated twice. into Geneious, and a multiple sequence alignment was The ratio of the radial growth of the pathogen to the performed using CLUSTALw (Kearse et al., 2012) of all radial growth of each unknown fungus was computed for ITS sequences as well as of the one sequence from L. each plate. These ratios were modeled with a linear mixed arvalis, and each of the consensus sequences from the model for repeated measures. Fixed effects of the model two unknown fungi (R47 and Hall). Sequences were nar- included the effects of pathogen, temperature, and day rowed to represent the diversity of the ITS region present (dap) as well as all possible interactions of these effects. in the database to include 12 sequences from R. zeae and Random effects included experiments and the interac- 12 sequences from R. oryzae (Table S1, Supporting Infor- tion between experiments, pathogen and temperature. mation). Additionally, the ITS region from one isolate of L. The covariance structure of the repeated measurements arvalis was used as an outgroup control in order to root the was modeled as compound symmetric. The same model tree (Table S1). Any ambiguously aligned sequences were was fit separately for each fungus, and for each set of manually trimmed and removed prior to phylogenetic experiments using SAS Proc Mixed (SAS Institute, ver- analysis. A neighbor-joining tree was built in Geneious sion 9.4, Cary, NC, USA). For each fungus, we compared (Kearse et al., 2012) using a Tamura and Nei’s distance the mean ratio (pathogen/fungus) of radial growth for model (Tamura & Nei, 1993), with 10 000 random repli- each pathogen, temperature, and day combination with cates, the bootstrap tree method, and selecting for a 70% the value of ‘one’. For each mean, we computed the P support threshold. value for the mean ratio to be less than one and adjusted the P value using the Sidak method (Westfall & SAS Insti- tute, 1999) for 75 multiple tests (three temperatures, five Pairing experiments to determine whether identified dates and five pathogens). fungi could inhibit growth of known sugar beet pathogens Pathogenicity testing of unknown fungi against All isolates were maintained as working cultures as sugar beet described above and kept at room temperature until used. Two pairing experiments were performed. The first tested Seeds of a universally susceptible sugar beet variety each unknown (R47 and Hall) or biocontrol fungus (Laet) ‘Monohikari’ (Nagata et al., 1991; Stump et al., 2004) with known sugar beet pathogens F19, Fob220a, R-9 and were planted into ‘yellow’ cone-tainers (4 cm in diam- R-1 (2 F. oxysporum f. sp. betae and 2 R. solani,respec- eter; Stuewe and Sons, Inc., Tangent, OR, USA) filled tively). The second tested each unknown or the known with steam pasteurised Fafard 2-SV potting mix (Sun L. arvalis-positive biocontrol fungus against Py54, WE41, Gro Horticulture, Agawam, MA, USA) and grown in a Phoma and Darnell (2 P. aphanidermatum and 2 Phoma greenhouse set at 24 ± 2/16∘C (daytime/nighttime) tem- spp., respectively). Using a 0.6-mm cork borer, each iso- perature, with a 18-h photoperiod, and 25–50% rela- late was transferred from working cultures to eight plates tive humidity for ∼ 6 weeks after sowing. Six weeks after of 2% water agar (WA) and incubated at 25∘Cinthedark sowing, plants were transferred to ‘black’ cone-tainers for 7 days, after which the WA plates were used to perform (7 cm in diameter, Stuewe and Sons, Inc., Tangent, OR, pairing experiments. Using a 0.6-mm cork borer, a plug USA) prefilled with pre-moistened (using tap water) pas- was removed from the WA cultures from each isolate and teurised potting mix and kept in the greenhouse using was placed ∼1.5 cm from the right side of a new Petri plate the same growing conditions as above until plants were also containing WA. A second plug from the same iso- at 8 weeks after sowing (approximately the sixth–eighth late was placed ∼1.5 cm on the left side of the same Petri true leaf stage). Four sources of artificial inoculum for plate directly across from the first plug. Then, using this each unknown fungus (R47 and Hall), as well as a same method, each unknown (R47 or Hall) or the known pathogenic R. solani isolate (R-9), were prepared using biocontrol fungus (Laet) was paired with each sugar beet a modified protocol originally described by Pierson & pathogen (F19, Fob220a, R-9, R-1, Py54, WE41, Phoma, Gaskill (1961). Briefly, 200 g of hulless barley grain and Darnell) as individual treatments. Control plates were (Bob’s Red Mill, Milwaukie, OR, USA; T. Vahger, per- also prepared by pairing each unknown or biocontrol sonal communication) was soaked in beakers overnight fungus with itself. Twelve WA plates per treatment were with distilled water (water was poured off to within

Table 1 Biological control studies (2006) in western Nebraska using unknown fungal isolates to manage multiple root pathogens of sugar beet comparing performance of cultivars with varying disease resistance packages

Entrya Disease Indexb Sugar (%) Root Yield (Metric Tons) Sugar Yield (kg ha−1) Sugar Loss to Molasses

Betaseed 8400/untreated 2.30 14.9 35.3 6224.7 2.10 Betaseed 8400/Hall 1.80 16.2 50.8 8220.2 1.90 Betaseed 8400/Laet 1.90 15.8 47.5 7758.5 1.90 Betaseed 8400/R47 1.90 15.9 45.9 7393.5 1.80 Betaseed 4546/untreated 2.70 16.4 28.7 4737.3 2.00 Ranger/untreated 2.80 15.7 17.5 2706.5 1.70 Betaseed 4595/untreated 1.30 17.9 49.3 9573.5 1.60 HM 2779 RZ/untreated 1.40 17.2 45.3 7843.4 1.50 HM 7172/untreated 1.40 16.4 52.2 9287.7 1.70 Betaseed 7341/untreated 1.90 17.6 54.0 10233.1 1.50 Betaseed 7310/untreated 1.60 17.2 54.4 10335.1 1.80 Monohikari/untreated 3.40 14.6 12.3 1925.1 2.10 HM 7235 RZ/untreated 1.40 18.0 49.7 9753.2 1.30 Betaseed 4100/untreated 2.00 16.1 38.5 5571.8 2.10 LSD 0.60 1.6 12.0 1831.2 0.34

LSD, least significant difference aSugar beet cultivar followed by fungal treatment. bDisease index, root disease index calculated as a weighted average of roots rated individually at harvest on a 0–4 scale with 0 = healthy root and 4 = completely rotted root. Disease index was then calculated by the following equation, DI = (DR1 × 1 + DR2 × 2 + DR3 × 3 + DR4 × 4)/(sum DR0–4). DR is the number of roots given the indicated disease rating. approximately 1 cm below the grain surface after soak- SAS Proc Npar1way (SAS Institute, version 9.4, Cary, NC, ing) and autoclaved twice for 45 min at 25 psi each time USA) with the Dwass, Steel, Critchlow-Fligner (DSCF) and then allowed to cool overnight. Agar plugs (7 mm option (Dwass, 1960; Steel, 1960; Critchlow & Fligner, diameter) from PDA cultures were placed approximately 1991) in order to test the pathogenicity of the treatments 1 cm deep in the autoclaved barley with two plugs per (Hall, R47, R-9 and Control). 200 g of barley and incubated for 17 days at 25∘Cand 16-h photoperiod prior to inoculation. Infested barley was Field data/yield experiments removed from beakers, air-dried for 3–4 days, and then ground in a Waring blender (Waring Laboratory, Torring- Field studies were conducted between 2006 and 2009 in ton, CT, USA) for 5 min, sterilising the blades with 70% research plots at the Panhandle Research and Extension ethanol between each isolate. As a negative control, 200 g Center in Scottsbluff, NE, USA, which was naturally of hulless barley grain was prepared as described above infested with multiple root pathogens including Beet but was left uninoculated and allowed to dry for 3–4 days necrotic yellow vein virus, R. solani, Pythium spp., Fusar- and then ground in a Waring blender. ium spp. and Phoma betae (R.M. Harveson, data not For pathogenicity testing, five sugar beet plants were shown). Studies in all years were planted in mid-May inoculated per inoculum source at 8 weeks after sowing with plots consisting of four rows, 55 cm in width and by placing approximately 0.5 g of each ground inocu- 12 m long. Plots were watered by overhead sprinkler lum (or sterile ground barley as the negative control) in irrigation (3.5–4.0 cm wk−1) and combined rainfall the soil next to the tap root (about 2 cm deep) of each approximating 20 cm, resulting in a total of at least 50-cm sugar beet plant. Plants were maintained in the green- moisture for all seasons. house in a completely randomised design by replicate During field studies in 2006, the two unknown fungal (inoculum source) and maintained at approximately 28∘C isolates (Hall and R47) as well as the positive mycopara- (16-h photoperiod) and soil was kept continually moist- sitic control (Laet) were used as in furrow treatments at ened. Four weeks after inoculation, roots were harvested, planting using a Rhizomania-resistant, but root rot sus- washed free of soil and examined for necrotic lesions or ceptible sugar beet cultivar ‘BetaSeed 8400’ (Betaseed, other root rot symptoms and scored on a scale of 0 (no Shakopee, MN, USA) and compared with additional sugar damage) to 7 (dead plant with root completely rotted) beet cultivars each varying in their disease tolerance (Ruppel & Hecker, 1982). Multiple comparisons of mean packages. Six replications of each treatment were com- rankings of lesion ratings among treatments were con- pletely randomised in blocks within field plots. During ducted based on the Wilcoxon Rank Sum test using the the remaining three seasons (2007 through 2009), the

Ann Appl Biol (2015) 5 © 2015 Association of Applied Biologists Identification and characterisation of BCA fungi to control sugar beet pathogens K.M. Webb et al. same three biological control isolates (Hall, R47 and Laet) yield parameters were determined at the Western Sugar were used singly, and in combination as treatments, but Cooperative Factory, Scottsbluff, NE, USA. Statistical only with the single cultivar ‘BetaSeed 8400’. Fungal analyses were conducted as a randomised complete block treatments consisted of colonising sugar beet seed (in the design with an analysis of variance (ANOVA) using SAS same manner as the autoclaved barley kernels) by filling (SAS Institute, version 4.3). Petri plates with moist autoclaved sugar beet seeds and then adding three 7-mm agar plug of each antagonistic Results fungus (R47, Hall and Laet) per plate. Petri plates were sealed with parafilm and incubated at 25∘C for several Isolation from field soils and identification through weeks until the fungi had grown throughout the plates morphology and ITS sequencing and colonised the autoclaved seed. Colonised seed was The two new isolates (R47 and Hall) used in this study then removed from plates, dried in plastic weigh boats were found in association with infected seedlings from overnight in a laminar flow hood, separated, and main- soil samples collected from two sugar beet fields from ∘ tained in vials or zip-lock bags at 4 C until used. An equal Box Butte County, NE, USA, a result of the root dis- quantity of colonised seeds was then combined with live ease diagnostic assay described earlier. Both isolates had seed of each treatment (3 g of each per seed packet per septate mycelium that was white (to buff) with regular, row for every plot) and planted. small, ball-shaped, reddish-brown coloured sclerotia that After stand establishment, disease counts were made ramified throughout the media. Hyphae in both isolates from one of the two outside rows at least four times were multinucleate and no hyphal clamp connections during each season based on dead or diseased plants were present in cultures, nor were any fruiting structures exhibiting wilting, interveinal chlorosis, or yellowed, or spores of any kind. Based on these morphological necrotic foliage, all of which are characteristic of characters, we determined that these isolates were not soilborne root diseases (Stewart, 1931; Kreutzer & similar to binucleate Rhizoctonia spp. Owing to shared Durrell, 1938, Ruppel, 1991; Harveson et al., 2009). The morphological characteristics with multinucleate Rhizoc- two middle rows of each plot were harvested by hand tonia spp. and the lack of hyphal clamps, we placed these in early- to mid-October of each year, and these same isolates as belonging to either the Ceratobasidiaceae or roots were assigned a root disease severity rating (0–4) R. solani (teleomorph Thanatephorus) (Sneh et al., 1991; based on disease symptoms present and on size and Sneh, 1996). The L. arvalis (teleomorph Isaria fuciformis) degree of root rot severity. For Rhizoctonia crown and isolate, in comparison, produced rapidly growing, pink, root rot or Phoma root rot, symptoms 0 = no disease, septate mycelium with numerous clamp connections 1 = small localised lesions with as much as 25% of root (Burdsall et al., 1980). Irregular-shaped pink sclero- surface affected, 2 = lesions coalescing with 26–50% of tia additionally formed on the agar surface without root affected, 3 = 51–75% of root surface covered with penetrating into the media. lesions, but no internal discoloration, and 4 = more than The internal transcribed spacer (ITS) region (∼700 bp) 75% of root surface covered with lesions and inter- was amplified from both unknown isolates (R47 and Hall) nal discoloration and penetration present (Harveson & using the ITS1 and ITS4 primers as previously reported Rush, 2002). For Fusarium yellows, symptoms 0 = no (White et al., 1990). For each isolate, PCR amplicons disease, 1 = less than 25% of vascular elements necrotic were sequenced, manually edited, and a consensus or localised lesions on root, 2 = 26–50% vascular necrosis sequence developed for each isolate. The consensus or less that 10% of taproot rotted, 3 = over 50% necrosis ITS sequence was about 447 bp for R47 and 648 bp for of vascular elements and 10–25% of taproot rotted, Hall, respectively. The consensus ITS sequence for each 4 = more than 25% of taproot rotted (Harveson & Rush, isolate (R47: KJ623714 and Hall: KJ623715) have been 1994, 2002). A disease index (DI) was then calculated submitted to GenBank (submission no. 1710040). A taking into account that different plots contained differ- BLASTn search on the consensus sequence from the ITS ent numbers of roots. The equation to calculate DI was as region showed that the sequence from isolate R47 had follows: DI = (DR1 × 1 + DR2 × 2 + DR3 × 3 + DR4 × 4)/the 98.4% pairwise identity and 96.5% identical sites to the sum of (DR0–DR4) with DR0 = number of roots rated a 0, nucleotide sequence of W. circinata (anamorph Rhizoctonia DR1 = number of roots rated a 1, etc. (Harveson & Rush, spp.) (GenBank accession no. HQ185366; Bit Score: 2002). For the situations where multiple pathogens were 728.044, E value = 0) (Fig. 1). The consensus sequence found that may confound the rating system, the higher from Hall had 99.8% pairwise identity, 99.2% identical (more severe) rating values were used. Additionally, sites to the nucleotide sequence of W. circinata (anamorph standard yield parameters were collected, including root Rhizoctonia spp.) [GenBank accession no. HQ270160; and sucrose yields and sugar loss to molasses (SLM). The Bit score: 1157.25, E value = 0 (Fig. 1)]. Based on the

Figure 1 Alignment of the consensus internal transcribed spacer (ITS) sequence from each unknown fungal isolate (A) Hall and (B) R47 aligned to most likely GenBank accession using BLASTn in Geneious. Highlighted letters indicate sequence differences between the two aligned ITS sequences. genetic similarity of the ITS gene sequences to isolates analysis we have putatively identiﬁed both unknown of Rhizoctonia spp., we performed a phylogenetic analysis fungi (R47 and Hall) as R. zeae for future associations. on the ITS regions using representative isolates of R. oryzae and R. zeae (Table S1). Based on this analysis, Ability of R47 and Hall to reduce growth of known both R47 and Hall cluster more similarly to isolates of sugar beet pathogens R. zeae (Fig. 2). Owing to morphological characteristics, To determine whether R47 and Hall are antagonistic to sequence similarity to Rhizoctonia spp., and phylogenetic known pathogens of sugar beet, we paired each isolate

the radial growth of R. solani isolate R-1 at 20∘C (Fig. 3). In contrast, R47 was quite variable in its ability to con- sistently reduce the radial growth of R. solani until later evaluation dates (Fig. 3). For example, R47 was unable to inhibit R. solani at 20∘Cor25∘C but was able to inhibit both R-1 and R-9 by 9 dap at 30∘C. R47 was able to effectively reduce the radial growth of both isolates of F. oxysporum f. sp. betae (Fig. 3), both isolates of P. aphanidermatum, and both isolates of Phoma spp at the time points tested (Fig. 4).

Pathogenicity testing on sugar beet To confirm that both unknown fungi were not pathogenic to sugar beet (as they had originally been isolated in association with known sugar beet pathogens), we performed pathogenicity testing in greenhouse experiments. Here, we compared pathogenicity using a known pathogenic isolate of R. solani (R-9), as well as a non-inoculated negative control (Fig. 5). R-9 was able to cause characteristic symptoms of Rhizoctonia root rot on the susceptible sugar beet cultivar ‘Monohikari’ (Nagata et al., 1991; Stump et al., 2004), which resulted in significantly higher disease ratings than the non-inoculated control. The mean ratings for both unknown fungi were not significantly Figure 2 Phylogenetic alignment of the internal transcribed spacer (ITS) region from each unknown fungal isolate (R47 and Hall) with representative different from the mean rating of the non-inoculated ITS regions from R. zeae and R. oryzae. All gene sequences were obtained control (Fig. 5). Neither the non-inoculated control, from GenBank using BLASTn. Only bootstrap scores of >70 are shown. L. nor either unknown fungal isolate caused symptoms arvalis (GenBank #EU622841.1) was used as an outgroup control for analysis. that were characteristic of Rhizoctonia root rot (wilting and/or dark, circular to oval root lesions; Fig. 5) or were with representative isolates of F. oxysporum f. sp. betae, any type of lesions visualised on roots (data not shown). R. solani, P. aphanidermatum,andPhoma spp. The ratio This indicates that the unknown fungal isolates were of radial mycelial growth of each pathogen to the radial non-pathogenic to a universally susceptible sugar beet variety. growth of each unknown (mean ratios were compared to a control value of one) was calculated to evaluate whether either isolate would reduce the radial growth of Field data/yield experiments to determine efficacy each pathogen at different temperatures. In general, both of fungi to reduce levels of disease in sugar beet unknown fungi were able to limit the radial growth of the The field location where the studies were conducted was pathogenic isolates of F. oxysporum f. sp. betae, P. aphani- initially designed to serve as a root disease nursery, and dermatum,andPhoma spp. whereas they were variable in thus every attempt had been made to purposefully cre- limiting the growth of R. solani (Figs 3 and 4). The L. arvalis ate and maintain high pathogen populations within these (Laet) isolate was able to inhibit both isolates of F. oxys- plots by growing continuous crops of sugar beets and leav- porum f. sp. betae, both isolates of Phoma spp.,andboth ing root residues on the soil surface with minimal tillage isolates of P. aphanidermatum (Figs 3 and 4); however, at practices. The first two studies (2006–2007) were sequen- some temperatures tested, control did not occur until later tially grown in the same place following at least three con- evaluation dates. Laet failed to reduce the radial growth of tinuous years with no rotation. No treatment differences R. solani isolate R-9 at any temperature tested; however, were observed for the 2007 season due in large part to it was able to limit the growth of R. solani isolate R-1 at high disease severity based on the fact that disease sever- 30∘C (Fig. 3). ity was the same for all treatments and root yields for all Hall generally had greater ability to reduce the growth treatments were in the range of 2–3 tons acre–1 (data not of pathogenic isolates than R47 because it was able to shown) and are therefore not included in this report. Fur- inhibit all isolates at each temperature evaluated (Figs 3 thermore, because of the poor results in 2007, the remain- and 4) except in the case where Hall was not able to limit ing two studies (2008–2009) were also carried out back

Figure 3 Effectiveness of unknown fungi (R47, Hall) to limit the radial growth of known sugar beet fungal pathogens Fusarium oxysporum f. sp. betae (F19 and Fob220a) and Rhizoctonia solani (R-1 and R-9). Ratio of pathogen radial growth to unknown fungus radial growth is shown at three temperatures: Top (20∘C), Middle (25∘C), and Bottom (30∘C). Dashed line indicates mean pathogen to fungus ratios from 4 plates per temperature treatment that are statistically less than ‘one’ at P value < 0.05 using a linear mixed model for repeated measures. Laetesaria arvalis isolate ‘Laet’ was used as a known mycoparasitic positive control. to back but were rotated to a different location within the During 2008, treatment with the R47 isolate resulted same field, preceded by a dry bean and sunflower crop in in statistically significant increases in several yield com- sequential years. ponents, compared with the same cultivar (BetaSeed During the 2006 study, the use of each unknown 8400) without treatment (Tables 2). Sucrose improve- fungus (R47 and Hall) resulted in improved root and ments during 2008 approached 2500 kg ha−1 (Table 2). −1 sucrose yields (almost 15 metric tons and 2000 kg ha , Similar increases were also observed in root yields, dis- respectively) when compared with Beta8400 that was left eased plants during the season, and disease severity untreated. Although these data were very encouraging, ratings at harvest (Table 2). The data from 2009 also indi- the disease and yield parameters were not all significantly cated significantly higher sucrose yield and disease resis- different (Table 1). Despite the lack of significant differ- tance performance for the treatment using R47 when ences in disease severity, at least one of the treatments compared with the same cultivar with no potential bio- (Betaseed 8400/Hall) achieved yields that were compa- rable with the performance of several cultivars that pos- control treatment (Table 3). No improvements were ever sessed multiple disease resistance packages against the seen with the treatment which included all three fungi same root pathogens such as Beta 7310, HM 7172 and combined because this treatment was not statistically HM 7235 RZ (all left untreated) all of which had less different from the untreated control and was worse disease than the susceptible Betaseed 8400 untreated con- than each of the three fungal treatments individually trol (Table 1). (Tables 2 and 3).

Figure 4 Effectiveness of unknown fungi (R47, Hall) to limit the radial growth of known sugar beet soilborne pathogens Phoma spp. (Darnell and Phoma) and Pythium aphanidermatum (Py54 and WE41). Ratio of pathogen radial growth to fungus radial growth is shown at three temperatures: top (20∘C), middle (25∘C) and bottom (30∘C). Dashed line indicates mean pathogen to fungus ratios from 4 plates per temperature treatment that are statistically less than ‘one’ at P value < 0.05 using the linear mixed model for repeated measures. Laetesaria arvalis isolate ‘Laet’ was used as a known mycoparasitic positive control.

Discussion for genus-level identification of fungi including Rhizoc- tonia spp. (Weiland & Sundsbak, 2000; Kõljalg et al., During screenings of soil samples from sugar beet pro- 2013) and to characterise populations to the species level duction sites and using sugar beet seedlings as a bait (Sharon et al., 2006; Okubara et al., 2008). Based on host, a number of unrecognised fungi commonly were characters described above, we identified these isolates found interspersed in cultures with several known sugar to be similar to Rhizoctonia spp. (Stalpers & Andersen, beet pathogens. Two of these unrecognised fungi (R47 1996). After sequence analysis of the ITS region from our and Hall) were isolated and purified from cultures, two unknown fungi, a close relationship was revealed and transferred to culture media for morphological and between both R47 and Hall with Rhizoctonia spp. (W. circi- molecular identification. Both isolates had septate, multi- nata Warcup & Talbot; Warcup & Talbot, 1962) which can nucleate mycelium that was white (to buff) with regular, be classified into two species, R. zeae Voorhees (Voorhees, small, ball-shaped reddish brown-coloured sclerotia 1934; Oniki et al., 1985) and R. oryzae Ryker & Gooch that ramified throughout the media. No hyphal clamp (Ryker & Gooch, 1938). Typically these two species can connections were present in cultures, nor were any be separated based on colony morphology with R. oryzae fruiting structures or spores of any kind observed. For having orange to salmon globose sclerotia and R. zeae molecular identification, we used the nuclear ribosomal having orange to brown, regular shaped sclerotia (Leiner ITS, which has long been used as a molecular marker & Carling, 1994; Stalpers & Andersen, 1996). Further

& Kuznia, 1994), some isolates previously have been shown to reduce plant diseases caused by R. solani such as stem canker and black scurf in potato (Brewer & Larkin, 2005). In that study, the R. zeae isolates were tested for pathogenicity on potato, as well as host crops such as barley and ryegrass and were found not to be pathogenic. Brewer & Larkin (2005) speculated that biocontrol of R. solani may be due to the ability of R. zeae isolates to outcompete isolates of R. solani when colonising host roots, and therefore prevent pathogenic R. solani from causing disease. Effective biocontrol agents have several modes of action for plant diseases, including antagonism, parasitism, competition for nutrients and/or colonization of plant tissues, antibiosis, etc. (Alabouvette et al., 2009). For example some secrete hyphal wall-degrading enzymes, causing death of the host cell, and finally utilise nutrients released through this process (Jeffries & Younger, 1994). Other biotrophic mycoparasites obtain their nutrition Figure 5 Pathogenicity of unknown fungi (Hall and R47) against suscepti- from living host cells (Jeffries & Younger, 1994). The ble sugar beet cultivar ‘Monohikari’. Unin, non-inoculated barley negative mechanism of antagonism (and/or perhaps mycopar- control; R-9, Rhizoctonia solani pathogenic isolate. Letters indicate statistical differences using the Kruskal–Wallis rank sum test of the ranked means asitism) for the reported isolates was not tested and of the disease severity rating scale. Disease severity was ranked 4 weeks after therefore is unknown, and future studies to determine inoculation on a scale of 0 (no disease) to 7 (plants completely dead) from five potential modes of action for these isolates should be per- sugar beet plants for each isolate. formed to fully understand their potential as a biological control. phylogenetic analysis indicated that both of our isolates Field evaluations using the two unknown fungi from clustered more similarly to isolates of R. zeae; therefore, three of the four years provided encouraging results for we have putatively identified these isolates as R. zeae. further work with these as potential biocontrol agents. However, future work will be needed to confirm this The fact that the two newly found fungi (R47 and Hall) identification, and we are currently investigating these increased yield and reduced disease, and did so as well as relationships with additional pathogenicity testing and the previously established biocontrol agent, L. arvalis,fur- molecular characterisation using other markers. ther illustrates their potential as biocontrol agents, thus R. zeae has been reported to be a pathogen of turf- making them attractive candidates for further study. They grass (Martin & Lucas, 1983; Burpee & Martin, 1992) appear to possess a large antagonistic host range, are and corn (Voorhees, 1934; Sumner & Bell, 1982), and largely unspecialised and capable of affecting many differ- R. oryzae has been shown to be a pathogen of rice (Ryker & ent diverse and unrelated pathogens. The ability to inhibit Gooch, 1938). Some isolates of R. zeae have been shown or outcompete many different soilborne pathogens also to be pathogens of sugar beet, but these tests were car- highlights a potential advantage if ever used on a larger ried out at the seedling stage and no discoloration was production scale. If incorporated as a seed treatment, observed on the roots (Windels & Kuznia, 1994). In our these could replace the need for multiple fungicidal seed studies, neither isolate caused symptoms of disease during treatments. For example, to provide protection against pathogenicity testing on older sugar beet (sixth–eighth R. solani as well as any of the Pythium species simultane- leaf stage), nor at the seedling stage (data not shown). ously, it would require the use of two separate fungicide However, we did not test pathogenicity of these two iso- classes (Mueller et al., 2013). Utilising these two isolates lates on other potential hosts that may be grown in rota- in an overall integrated management strategy combined tion with sugar beet. This is another aspect that we will with commercial fungicides and/or other cultural prac- examine in future experiments to determine whether tices should be investigated more thoroughly in future they could serve as potential risks to other crops in the experiments. Interestingly, in this study, we saw another rotation before they can be used as part of a practical man- potential benefit to using these organisms at planting. In agement strategy for root diseases in sugar beet. addition to reducing stand loss, the data also suggested However, although R. zeae can be a pathogen of some that season-long protection from multiple root diseases hosts (Voorhees, 1934; Burpee & Martin, 1992; Windels was provided (Tables 1, 2, and 3). We also saw that when

Table 2 Biological control ﬁeld (2008) in western Nebraska studies using unknown fungal isolates to manage multiple root pathogens of sugar beet

Entrya Diseased Plantsb Disease Indexc Sugar (%) Root Yield (Metric Tons) Sugar Yield (kg ha−1) Sugar Loss to Molasses

Betaseed 8400/untreated 91.8a 2.40a 12.9 26.2 dc 3434c 3.10 Betaseed 8400/R47 54.0b 1.80bc 13.2 45.3a 5877a 2.90 Betaseed 8400/Laet 50.8b 1.40c 15.3 35.8b 5937a 2.80 Betaseed 8400/Hall 54.6b 1.50c 13.5 39.0ab 5105bc 3.10 Betaseed 8400/all three combinedd 105.2a 2.30ab 13.9 23.3d 4112bc 3.20 aSugar beet cultivar followed by fungal treatment. bTotal number of diseased plants identiﬁed within plots. cDisease index = root disease index: a weighted average of roots rated individually at harvest on a 0–4 scale with 0 = healthy root and 4 = completely rotted root. Disease index was then calculated by the following equation: DI = (DR1 × 1 + DR2 × 2 + DR3 × 3 + DR4 × 4)/(sum DR0–4). DR is the number of roots given the indicated disease rating. dMycoparasite treatment for all three combined included each mycoparasitic isolate (R47, Laet, and Hall). DR is the number of roots given the indicated disease rating. Means within columns followed by different letters are statistically different according to Fisher’s protected least signiﬁcant difference test (P > 0.05).

Table 3 Biological control ﬁeld studies (2009) in western Nebraska using unknown fungal isolates to manage multiple root pathogens of sugar beet

Entrya Diseased Plantsb Disease Indexc Sugar (%) Root Yield (Tons) Sugar Yield (lb a−1) Sugar Loss to Molasses

Betaseed 8400/Untreated 22.6a 3.64 11.2 6.2c 1500.0c 2.12b Betaseed 8400/R47 12.3b 2.91 13.1 15.3a 5064.6a 1.92a Betaseed 8400/Laet 7.7c 2.86 13.2 12.5ab 3973.7ab 2.01b Betaseed 8400/Hall 11.0b 3.03 12.0 11.6ab 3497.3ab 2.05b Betaseed 8400/All three combinedd 15.2b 3.50 11.8 9.6bc 3039.2b 2.09b aSugar beet cultivar followed by fungal treatment. bTotal number of diseased plants identified within plots. cDisease index = root disease index: a weighted average of roots rated individually at harvest on a 0–4 scale with 0 = healthy root and 4 = completely rotted root. Disease index was then calculated by the following equation: DI = (DR1 × 1 + DR2 × 2 + DR3 × 3 + DR4 × 4)/(sum DR0–4). DR = number of roots given the indicated disease rating. dMycoparasite treatment for all three combined included each mycoparasitic isolate (R47, Laet and Hall). Means within columns followed by different letters are statistically different according to Fisher’s protected least significant difference test (P > 0.05). all three fungi (R47, Hall, and L. arvalis)wereusedas Averre, 1997). This phenomenon has been created often a single treatment, there were no statistically significant through specific agronomic management practices (i.e. differences in disease realised when compared with the cropping monoculture, reviewed in the study by Weller untreated control (Tables 2 and 3). It is possible that when et al., 2002; Mazzola, 2004). Biological control strategies all three fungi were combined that they ended up antag- using the concept of suppressive soils have been recog- onistic to each other and/or were unable to outcompete nised for well over 100 years, but the complex nature the pathogens tested for resources in these treatments. of disease-suppressive soils has yet to be elucidated fully The successful implementation of biological control in for all known suppressive pathosystems (Mazzola, 2004; many cropping systems has been quite difficult, particu- Weller et al., 2002). In most systems, the mechanism of larly using introduced fungi, primarily because of fungi- control is poorly understood; however, for most disease stasis, competition, prevention of antibiosis, etc. (Lewis suppressive soils, it is believed that disease reduction & Papavizas, 1991; Xu et al., 2011). Therefore, prior to primarily can be explained by microbial antagonism making recommendations on how to utilise these fungi exerted on the pathogen(s) by antibiosis, competition in sugar beet production systems, additional studies on within the rhizosphere, and parasitism and/or predation mode of biocontrol activity, best product formulation, (Weller et al., 2002). most effective delivery system(s), overall field efficacy, and impacts on entire crop rotation systems need to be Acknowledgements performed and optimised. Several potential biocontrol agents have been The authors would like to thank Clay Carlson, Bob identified from soil ecosystems that have been shown Hawley, and Kathy Nielsen for lab, field and green- to limit the development of some soilborne diseases, house assistance. They also thank Addison Reed and often termed ‘disease-suppressive soils’ (Shurtleff & Claire Freeman for their assistance in DNA extractions,

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